Ice-based lipid nanoparticle formulation for delivery of mRNA

ABSTRACT

The present invention provides, among other things, compositions and methods of formulating nucleic acid-containing nanoparticles comprising no more than three distinct lipids components, one distinct lipid component being a sterol-based cationic lipid. In some embodiments, the present invention provides compositions and methods in which the lipid nanoparticles further comprise helper lipids and PEG-modified lipids. The resulting formulation comprises a high encapsulation percentage for nucleic acids.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/420,421, filed Nov. 10, 2016, U.S. Provisional Application Ser.No. 62/420,428, filed Nov. 10, 2016, U.S. Provisional Application Ser.No. 62/421,021, filed Nov. 11, 2016, U.S. Provisional Application Ser.No. 62/421,007, filed Nov. 11, 2016, U.S. Provisional Application Ser.No. 62/464,327, filed Feb. 27, 2017, and U.S. Provisional ApplicationSer. No. 62/464,330, filed Feb. 27, 2017, the disclosures of which arehereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “MRT-1247US_SL” on Nov.10, 2017). The .txt file was generated Nov. 10, 2017 and is 57,411 bytesin size. The entire contents of the Sequence Listing are hereinincorporated by reference.

BACKGROUND

Nucleic acid-based technologies are increasingly important for varioustherapeutic applications including, but not limited to, messenger RNAtherapy. Efforts to deliver nucleic acids have included the creation ofcompositions formulated to protect nucleic acids from degradation whendelivered in vivo. One type of delivery vehicle for nucleic acids hasbeen lipid nanoparticles. Important parameters to consider for thesuccessful use of lipid nanoparticles as a delivery vehicle includelipid nanoparticle formation, physical properties of lipid components,nucleic acid encapsulation efficiencies, in vivo nucleic acid releasepotential, and lipid nanoparticle toxicity.

SUMMARY OF THE INVENTION

This present invention offers a unique solution wherein sterol-basedcationic lipid, helper lipid and PEG-modified lipid form a lipidnanoparticle formulation of RNA. This inventive three lipid componentsystem upon formulation shows high RNA encapsulation efficiencies andsuccessful efficacious delivery in vivo, particularly pulmonarydelivery. Such formulation systems offer additional advantages of lowerlipid load (compared to other conventional four lipid component systems)and higher tolerability/lower toxicity as the metabolized products ofthe cationic lipid (cholesterol derivative lipid) is cholesterol.

The invention is based, in part, on the surprising discovery that athree lipid component system based on sterol-based cationic lipids isunexpectedly effective in delivering mRNA and producing encoded proteinor peptide in vivo, particularly in the lung. Indeed, prior to thepresent invention, cationic lipids have been extensively explored as animportant component of liposomes used to encapsulate nucleic acids,including mRNA, for in vivo delivery. Due to the uniquely fragile andlong structure of mRNA and the complicated in vivo translation process,cationic lipids used in the liposomes typically play two roles. First,cationic lipids promote interaction with negatively charged mRNA duringencapsulation, circulation and endocytosis, thereby capturing andprotecting the mRNA. Then, once inside cytosol, cationic lipids need tobe able to release the mRNA so that the mRNA can be translated toproduce the encoded protein or peptide. Some cationic lipids, inparticular, known as titratable cationic lipids, are particularlyeffective in delivering mRNA. Surprisingly, the present inventors foundthat liposomes comprising the sterol-based cationic lipids describedherein can have an even higher encapsulation percentage for mRNA and canbe even more effective in delivering various mRNA in vivo. Particularly,liposomes comprising the sterol-based cationic lipids described hereincan be incredibly effective for pulmonary delivery of mRNA, andsurprisingly successful for delivering mRNA via nebulization. Thus, thepresent inventors have demonstrated that the three lipid componentsystem can be uniquely useful in delivering mRNA for highly efficientand sustained production of protein or peptide (e.g., therapeuticprotein) in vivo, particularly in the pulmonary system. The presentinvention therefore permits an improved mRNA therapy that cansignificantly reduce the required amount of mRNA and associated lipids,administration frequency, and possible side effects, providing safer,more potent, and patient friendly mRNA therapy for various diseases.

In one aspect, the present invention provides methods of deliveringnucleic acids in vivo comprising administering by pulmonary delivery toa subject in need of delivery a composition comprising nucleic acids;and lipid nanoparticles encapsulating the nucleic acids, wherein eachindividual lipid nanoparticle comprises no more than three distinctlipid components, one distinct lipid component being a sterol-basedcationic lipid.

In another aspect, the present invention provides methods of deliveringnucleic acids in vivo comprising administering by pulmonary delivery toa subject in need of delivery a composition comprising nucleic acids,wherein the nucleic acids encode a Cystic Fibrosis TransmembraneConductance Regulator (CFTR) protein; and lipid nanoparticlesencapsulating the nucleic acids, wherein each individual lipidnanoparticle comprises no more than three distinct lipid components, onedistinct lipid component being a sterol-based cationic lipid.

In some embodiments, the pulmonary delivery comprises nebulization. Insome embodiments, said subject is a subject in need of delivery.

In some embodiments, the lipid nanoparticles have an encapsulationpercentage for nucleic acids of at least 70% (e.g., at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%).

In some embodiments, the three distinct lipid components comprise one orboth of helper lipids and PEG-modified lipids.

In some embodiments, the sterol-based cationic lipid has a structureaccording to Formula (A),B-L¹-S  (Formula A),or a protonated form thereof, wherein B is a basic functional groupwherein the protonated form has a pK_(a) that is no more than about 8.0;L¹ is an optionally substituted linker group that is a C₁-C₂₀ alkyleneor a 2- to 20-membered heteroalkylene; and S is a sterol.

In some embodiments, B is an optionally substituted 5- or 6-memberednitrogen-containing heteroaryl. In some embodiments, B is a groupselected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,pyridyl, pyrimidyl, pyrazinyl, and pyridazinyl, each of which isoptionally substituted. In some embodiments, B is a group selected frompyrrolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl,pyrazinyl, and pyridazinyl, each of which is optionally substituted. Insome embodiments, B is a group selected from pyrrolyl, pyrazolyl,triazolyl, tetrazolyl, pyrimidyl, pyrazinyl, and pyridazinyl, each ofwhich is optionally substituted.

In some embodiments, L¹ is an optionally substituted linker group thatis a C₁-C₂₀ alkylene. In some embodiments, L¹ is an optionallysubstituted linker group that is a 2- to 20-membered heteroalkylene. Insome embodiments, L¹ is a 2- to 20-membered heteroalkylene that isnon-peptidic. In some embodiments, L¹ comprises a moiety that is anester group, an amide group, a carbamate group, a carbonate group, or aurea group. In some embodiments, L¹ comprises a moiety that is an amidegroup, a carbamate group, a carbonate group, or a urea group. In someembodiments, L¹ comprises a moiety that is an amide group, a carbonategroup, or a urea group. In some embodiments, L¹ is —X¹—C(X³)—X²,—(C₁-C₁₉ alkylene)-X¹—C(X³)—X², —X¹—C(X³)—X²(C₁-C₁₉ alkylene)-, —(C₁-C₁₉alkylene)-X¹—, —X¹—(C₁-C₁₉ alkylene)-, wherein each X¹ and X² isindependently, a covalent bond, —O—, —S—, or —NH—; X³ is independently═O, ═S, or ═NH; and wherein said C₁-C₁₉ alkylene is optionallysubstituted. In some embodiments, L¹ does not comprise substituentshaving the structure —N(R′)₂, or a positively charged form thereof,wherein each R′ is independently hydrogen or optionally substitutedC₁-C₂₀ alkyl.

In some embodiments, S is a zoosterol, or an oxidized or reduced formthereof. In some embodiments, S is a phytosterol, or an oxidized orreduced form thereof. In some embodiments, S is a synthetic sterol, oran oxidized or reduced form thereof. In some embodiments, S is a sterolselected from cholesterol, an oxidized form of cholesterol, a reducedform of cholesterol, alkyl lithocholate, stigmasterol, stigmastanol,campesterol, ergosterol, and sitosterol. In some embodiments, S is asterol selected from an oxidized form of cholesterol, a reduced form ofcholesterol, alkyl lithocholate, stigmasterol, stigmastanol,campesterol, ergosterol, and sitosterol. In some embodiments, S is asterol selected from

wherein R is optionally substituted C₁-C₂₀ alkyl. In some embodiments, Sis a sterol selected from

wherein R is optionally substituted C₁-C₂₀ alkyl.

In some embodiments, the sterol-based cationic lipid comprises imidazolecholesterol ester (ICE). In some embodiments, the sterol-based cationiclipid does not comprise imidazole cholesterol ester (ICE).

In some embodiments, the nucleic acids are selected from DNA, siRNA,microRNA, and/or mRNA. In some embodiments, the nucleic acids are mRNAencoding a protein or a peptide. In some embodiments, the mRNA encodinga protein or a peptide is codon-optimized. In some embodiments, the mRNAcomprises one or more modified nucleotides. In some embodiments, themRNA comprises a modification of the 5′ untranslated region of saidmRNA. In some embodiments, said modification of the 5′ untranslatedregion comprises the inclusion of a Cap1 structure. In some embodiments,the mRNA comprises a modification of the 3′ untranslated region of saidmRNA. In some embodiments, said modification of the 3′ untranslatedregion comprises the inclusion of a poly A tail.

In some embodiments, the lipid nanoparticles have a size less than about100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm,50 nm, 45 nm or 40 nm.

In another aspect, the present invention provides compositionsformulated for nebulization comprising nucleic acids; and lipidnanoparticles encapsulating the nucleic acids, wherein each individuallipid nanoparticle comprises no more than three distinct lipidcomponents, one distinct lipid component being a sterol-based cationiclipid, and further wherein the lipid nanoparticles have an encapsulationpercentage for nucleic acids of at least 70% (e.g., at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%).

In another aspect, the present invention provides compositionscomprising nucleic acids; and lipid nanoparticles encapsulating thenucleic acids, wherein each individual lipid nanoparticle comprises nomore than three distinct lipid components, one distinct lipid componentbeing a sterol-based cationic lipid, and further wherein the lipidnanoparticles have an encapsulation percentage for nucleic acids of atleast 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%).

In some embodiments, the lipid nanoparticles have an encapsulationpercentage for nucleic acids of at least 85%. In some embodiments, thelipid nanoparticles have an encapsulation percentage for nucleic acidsof at least 90%.

In another aspect, the present invention provides compositionscomprising mRNA encoding a Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) protein; and lipid nanoparticles encapsulating themRNA, wherein each individual lipid nanoparticle comprises no more thanthree distinct lipid components, one distinct lipid component being asterol-based cationic lipid, and further wherein the lipid nanoparticleshave an encapsulation percentage for mRNA of at least 80%.

In some embodiments, the lipid nanoparticles have an encapsulationpercentage for mRNA of at least 85% or at least 90%.

In another aspect, the present invention provides compositionscomprising nucleic acids; and lipid nanoparticles encapsulating thenucleic acids, wherein each individual lipid nanoparticle comprises nomore than three distinct lipid components, one distinct lipid componentbeing a sterol-based cationic lipid, and further wherein thesterol-based cationic lipid constitutes no more than 70% of the totallipids (e.g., no more than 68%, no more than 67%, no more than 66%, nomore than 65%, no more than 60%, no more than 55%, no more than 50%, nomore than 45%, or no more than 40%).

In another aspect, the present invention provides compositionscomprising mRNA encoding a Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) protein; and lipid nanoparticles encapsulating themRNA, wherein each individual lipid nanoparticle comprises no more thanthree distinct lipid components, one distinct lipid component being asterol-based cationic lipid, and further wherein the sterol-basedcationic lipid constitutes no more than 70% of the total lipids (e.g.,no more than 68%, no more than 67%, no more than 66%, no more than 65%,no more than 60%, no more than 55%, no more than 50%, no more than 45%,or no more than 40%).

In some embodiments, the sterol-based cationic lipid constitutes no morethan 65% of the total lipids. In some embodiments, the sterol-basedcationic lipid constitutes no more than 60% of the total lipids. In someembodiments, the three distinct lipid components comprise one or both ofhelper lipids and PEG-modified lipids.

In some embodiments, the sterol-based cationic lipid has a structureaccording to Formula (A),B-L¹-S  (Formula A),or a protonated form thereof, wherein B is a basic functional groupwherein the protonated form has a pK_(a) that is no more than about 8.0;L¹ is an optionally substituted linker group that is a C₁-C₂₀ alkyleneor a 2- to 20-membered heteroalkylene; and S is a sterol.

In some embodiments, B is an optionally substituted 5- or 6-memberednitrogen-containing heteroaryl. In some embodiments, B is a groupselected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,pyridyl, pyrimidyl, pyrazinyl, and pyridazinyl, each of which isoptionally substituted. In some embodiments, B is a group selected frompyrrolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl,pyrazinyl, and pyridazinyl, each of which is optionally substituted. Insome embodiments, B is a group selected from pyrrolyl, pyrazolyl,triazolyl, tetrazolyl, pyrimidyl, pyrazinyl, and pyridazinyl, each ofwhich is optionally substituted.

In some embodiments, L¹ is an optionally substituted linker group thatis a C₁-C₂₀ alkylene. In some embodiments, L¹ is an optionallysubstituted linker group that is a 2- to 20-membered heteroalkylene. Insome embodiments, L¹ is a 2- to 20-membered heteroalkylene that isnon-peptidic. In some embodiments, L¹ comprises a moiety that is anester group, an amide group, a carbamate group, a carbonate group, or aurea group. In some embodiments, L¹ comprises a moiety that is an amidegroup, a carbamate group, a carbonate group, or a urea group. In someembodiments, L¹ comprises a moiety that is an amide group, a carbonategroup, or a urea group. In some embodiments, L¹ is —X¹—C(X³)—X²,—(C₁-C₁₉ alkylene)-X¹—C(X³)—X², —X¹—C(X³)—X²(C₁-C₁₉ alkylene)-, —(C₁-C₁₉alkylene)-X¹—, —X¹—(C₁-C₁₉ alkylene)-, wherein each X¹ and X² isindependently, a covalent bond, —O—, —S—, or —NH—; X³ is independently═O, ═S, or ═NH; and wherein said C₁-C₁₉ alkylene is optionallysubstituted. In some embodiments, L¹ does not comprise substituentshaving the structure —N(R′)₂, or a positively charged form thereof,wherein each R′ is independently hydrogen or optionally substitutedC₁-C₂₀ alkyl.

In some embodiments, S is a zoosterol, or an oxidized or reduced formthereof. In some embodiments, S is a phytosterol, or an oxidized orreduced form thereof. In some embodiments, S is a synthetic sterol, oran oxidized or reduced form thereof. In some embodiments, S is a sterolselected from cholesterol, an oxidized form of cholesterol, a reducedform of cholesterol, alkyl lithocholate, stigmasterol, stigmastanol,campesterol, ergosterol, and sitosterol. In some embodiments, S is asterol selected from an oxidized form of cholesterol, a reduced form ofcholesterol, alkyl lithocholate, stigmasterol, stigmastanol,campesterol, ergosterol, and sitosterol. In some embodiments, S is asterol selected from

wherein R is optionally substituted C₁-C₂₀ alkyl. In some embodiments, Sis a sterol selected from

wherein R is optionally substituted C₁-C₂₀ alkyl.

In some embodiments, the sterol-based cationic lipid comprises imidazolecholesterol ester (ICE). In some embodiments, the sterol-based cationiclipid does not comprise imidazole cholesterol ester (ICE).

In some embodiments, the nucleic acids are selected from DNA, siRNA,microRNA, and/or mRNA. In some embodiments, the nucleic acids are mRNAencoding a protein or a peptide. In some embodiments, the mRNA encodinga protein or a peptide is codon-optimized. In some embodiments, the mRNAcomprises one or more modified nucleotides. In some embodiments, themRNA comprises a modification of the 5′ untranslated region of saidmRNA. In some embodiments, said modification of the 5′ untranslatedregion comprises the inclusion of a Cap1 structure. In some embodiments,the mRNA comprises a modification of the 3′ untranslated region of saidmRNA. In some embodiments, said modification of the 3′ untranslatedregion comprises the inclusion of a poly A tail.

In some embodiments, the lipid nanoparticle has a size less than about100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm,50 nm, 45 nm or 40 nm.

In some embodiments, the mRNA encoding a CFTR protein comprises SEQ IDNO: 1. In some embodiments, the mRNA encoding a CFTR protein comprises apolynucleotide sequence at least 70%, 75%, 80%, 85%, 90%, or 95%identical to SEQ ID NO: 1.

In some embodiments, the lipid nanoparticle is formed by mixing an mRNAsolution and a lipid solution into a 20% ethanol. In some embodiments,the lipid nanoparticles are further purified by Tangential FlowFiltration.

In another aspect, the present invention provides methods of deliveringmRNA in vivo comprising administering a composition to a subject. Insome embodiments, said subject is a subject in need of delivery. In someembodiments, the composition is administered intravenously. In someembodiments, the composition is administered by pulmonary delivery. Insome embodiments, the pulmonary delivery comprises nebulization.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this disclosure, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Both terms are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING

The drawings are for illustration purposes only, not for limitation.

FIG. 1 depicts exemplary immunohistochemical detection of human CysticFibrosis Transmembrane Conductance Regulator (hCFTR) protein in mouselung 24 hours after treatment with ICE-based LNPs encapsulatingcodon-optimized hCFTR mRNA, B=Untreated mouse lung at 10× & 20×magnification, respectively. C, D=CO-hCFTR mRNA ICE LNP-treated mouselung at 10× & 20× magnification, respectively.

FIG. 2 depicts exemplary immunohistochemical detection of human CFTRprotein in mouse lung 24 hours after treatment with ICE-based LNPsencapsulating codon-optimized hCFTR. A, B=Untreated mouse lung at 10× &20× magnification, respectively. C, D=CO-hCFTR mRNA ICE LNP-treatedmouse lung at 10× & 20× magnification, respectively.

FIG. 3 depicts exemplary immunohistochemical detection of human CysticFibrosis Transmembrane Conductance Regulator (hCFTR) protein in thelungs of rats 24 hours after treatment with nebulized ICE-based lipidnanoparticles (LNPs) (5% PEG) encapsulating codon-optimized hCFTR mRNA.

FIG. 4 depicts exemplary immunohistochemical detection of hCFTR proteinin the lungs of rats 24 hours after treatment with ICE-based LNPs (5%PEG) encapsulating codon-optimized hCFTR mRNA via intratrachealadministration.

FIG. 5 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of rats 24 hours after treatment with nebulizedICE-based LNPs (3% PEG) encapsulating codon-optimized (CO) hCFTR mRNA.

FIG. 6 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of rats 24 hours after treatment with ICE-basedLNPs (3% PEG) encapsulating CO-hCFTR mRNA via intratracheal(Microsprayer) administration.

FIG. 7 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of CFTR KO (knockout) mice 24 hours after treatmentwith nebulized ICE-based LNPs (N/P=2) encapsulating CO-hCFTR mRNA.

FIG. 8 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of CFTR KO mice 24 hours after treatment withnebulized ICE-based LNPs (N/P=4) encapsulating CO-hCFTR mRNA.

FIG. 9 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of wild-type mice 24 hours after treatment withnebulized ICE-based LNPs encapsulating various mRNA constructs includingCFTR (CO-hCFTR mRNA), STOP (nonsense mutated CO-hCFTR mRNA unable to betranslated into protein), and FFL (Firefly Luciferase mRNA).

FIG. 10 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of wild-type mice at various time points after asingle exposure to nebulized ICE-based LNPs encapsulating CO-hCFTR mRNA.

FIG. 11 depicts an exemplary graph of the ratio of copies of CO-hCFTRmRNA per micrograms of total RNA in frozen lung sections from ratstreated with either controls (buffer, Empty ICE LNP) or CO-hCFTR mRNAloaded ICE LNPs.

FIG. 12 depicts an exemplary graph of fold-increase of copies ofCO-hCFTR over endogenous levels of CFTR mRNA in frozen lung sections ofrats treated with either controls (buffer, Empty ICE LNP) or CO-hCFTRmRNA loaded ICE LNPs.

FIG. 13 depicts exemplary immunohistochemical detection of human CFTRprotein in the lungs of rats after a single exposure to various doses ofnebulized ICE-based LNPs encapsulating CO-hCFTR mRNA.

FIGS. 14A and 14B depict exemplary graphs of the effects of variousdosages of hCFTR mRNA formulated with either branched PEI (FIG. 14A) orICE (FIG. 14B) on the respiratory rate of rats relative to a control.

FIG. 15 depicts an exemplary graph showing a dose response ofchloride-ion channel activity induced by hCFTR mRNA.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

Alkyl: As used herein, “alkyl” refers to a radical of a straight-chainor branched saturated hydrocarbon group having from 1 to 15 carbon atoms(“C₁₋₁₅ alkyl”). In some embodiments, an alkyl group has 1 to 3 carbonatoms (“C₁₋₃ alkyl”). Examples of C₁₋₃ alkyl groups include methyl (C₁),ethyl (C₂), n-propyl (C₃), and isopropyl (C₃). In some embodiments, analkyl group has 8 to 12 carbon atoms (“C₈₋₁₂ alkyl”). Examples of C₈₋₁₂alkyl groups include, without limitation, n-octyl (C₈), n-nonyl (C₉),n-decyl (C₁₀), n-undecyl (C₁₁), n-dodecyl (C₁₂) and the like. The prefix“n-” (normal) refers to unbranched alkyl groups. For example, n-C₈ alkylrefers to —(CH₂)₇CH₃, n-C₁₀ alkyl refers to —(CH₂)₉CH₃, etc.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COHO. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxyl-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinis expressed and retained within the target tissue (also referred to as“local distribution” or “local delivery”), and situations in which anmRNA is delivered to a target tissue and the encoded protein isexpressed and secreted into patient's circulation system (e.g., serum)and systematically distributed and taken up by other tissues (alsoreferred to as “systemic distribution” or “systemic delivery).

Encapsulation: As used herein, the term “encapsulation,” or grammaticalequivalent, refers to the process of confining an mRNA molecule within ananoparticle.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a peptide, polypeptide, orprotein, assembly of multiple polypeptides (e.g., heavy chain or lightchain of antibody) into an intact protein (e.g., antibody) and/orpost-translational modification of a polypeptide or fully assembledprotein (e.g., antibody). In this application, the terms “expression”and “production,” and grammatical equivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required fora quantity such as nucleic acid or protein concentration or activity tofall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

Impurities: As used herein, the term “impurities” refers to substancesinside a confined amount of liquid, gas, or solid, which differ from thechemical composition of the target material or compound. Impurities arealso referred to as contaminants.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “localdistribution,” “local delivery,” or grammatical equivalent, refer totissue specific delivery or distribution. Typically, local distributionor delivery requires a protein or peptide encoded by mRNAs be translatedand expressed intracellularly or with limited secretion that avoidsentering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one peptide,polypeptide or protein. mRNA as used herein encompasses both modifiedand unmodified RNA. mRNA may contain one or more coding and non-codingregions. mRNA can be purified from natural sources, produced usingrecombinant expression systems and optionally purified, chemicallysynthesized, etc. Where appropriate, e.g., in the case of chemicallysynthesized molecules, mRNA can comprise nucleoside analogs such asanalogs having chemically modified bases or sugars, backbonemodifications, etc. An mRNA sequence is presented in the 5′ to 3′direction unless otherwise indicated. In some embodiments, an mRNA is orcomprises natural nucleosides (e.g., adenosine, guanosine, cytidine,uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine,inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine); chemically modified bases;biologically modified bases (e.g., methylated bases); intercalatedbases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose); and/or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

In some embodiments, the mRNA comprises one or more nonstandardnucleotide residues. The nonstandard nucleotide residues may include,e.g., 5-methyl-cytidine (“5mC”), pseudouridine (“ψU”), and/or2-thio-uridine (“2sU”). See, e.g., U.S. Pat. No. 8,278,036 orWO2011012316 for a discussion of such residues and their incorporationinto mRNA. The mRNA may be RNA, which is defined as RNA in which 25% ofU residues are 2-thio-uridine and 25% of C residues are5-methylcytidine. Teachings for the use of RNA are disclosed US PatentPublication US 2012/0195936 and international publication WO2011/012316, both of which are hereby incorporated by reference in theirentirety. The presence of nonstandard nucleotide residues may render anmRNA more stable and/or less immunogenic than a control mRNA with thesame sequence but containing only standard residues. In furtherembodiments, the mRNA may comprise one or more nonstandard nucleotideresidues chosen from isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurineand 2-chloro-6-aminopurine cytosine, as well as combinations of thesemodifications and other nucleobase modifications. Certain embodimentsmay further include additional modifications to the furanose ring ornucleobase. Additional modifications may include, for example, sugarmodifications or substitutions (e.g., one or more of a 2′-O-alkylmodification, a locked nucleic acid (LNA)). In some embodiments, theRNAs may be complexed or hybridized with additional polynucleotidesand/or peptide polynucleotides (PNA). In embodiments where the sugarmodification is a 2′-O-alkyl modification, such modification mayinclude, but are not limited to a 2′-deoxy-2′-fluoro modification, a2′-O-methyl modification, a 2′-O-methoxyethyl modification and a2′-deoxy modification. In certain embodiments, any of thesemodifications may be present in 0-100% of the nucleotides—for example,more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of theconstituent nucleotides individually or in combination.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA. Furthermore, theterms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleicacid analogs, i.e., analogs having other than a phosphodiester backbone.For example, the so-called “peptide nucleic acids,” which are known inthe art and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. Theterm “nucleotide sequence encoding an amino acid sequence” includes allnucleotide sequences that are degenerate versions of each other and/orencode the same amino acid sequence. Nucleotide sequences that encodeproteins and/or RNA may include introns. Nucleic acids can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated. In someembodiments, a nucleic acid is or comprises natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and2-thiocytidine); chemically modified bases; biologically modified bases(e.g., methylated bases); intercalated bases; modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/ormodified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages). In some embodiments, the presentinvention is specifically directed to “unmodified nucleic acids,”meaning nucleic acids (e.g., polynucleotides and residues, includingnucleotides and/or nucleosides) that have not been chemically modifiedin order to facilitate or achieve delivery.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post-natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable salt: Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge et al., describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or malonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium. quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequarternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder (e.g., cysticfibrosis) but may or may not display symptoms of the disease ordisorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Systemic distribution or delivery: As used herein, the terms “systemicdistribution,” “systemic delivery,” or grammatical equivalent, refer toa delivery or distribution mechanism or approach that affect the entirebody or an entire organism. Typically, systemic distribution or deliveryis accomplished via body's circulation system, e.g., blood stream.Compared to the definition of “local distribution or delivery.”

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Transfer vehicle: In some embodiments, the transfer vehicle is aliposomal vesicle, or other means to facilitate the transfer of anucleic acid to target cells and tissues. Suitable transfer vehiclesinclude, but are not limited to, liposomes, nanoliposomes,ceramide-containing nanoliposomes, proteoliposomes, nanoparticulates,calcium phosphor-silicate nanoparticulates, calcium phosphatenanoparticulates, silicon dioxide nanoparticulates, nanocrystallineparticulates, semiconductor nanoparticulates, poly(D-arginine),nanodendrimers, starch-based delivery systems, micelles, emulsions,niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers,peptides and other vectorial tags. Also contemplated is the use ofbionanocapsules and other viral capsid proteins assemblies as a suitabletransfer vehicle. (Hum. Gene Ther. 2008 September; 19(9):887-95).

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

Yield: As used herein, the term “yield” refers to the percentage of mRNArecovered after encapsulation as compared to the total mRNA as startingmaterial. In some embodiments, the term “recovery” is usedinterchangeably with the term “yield”.

DETAILED DESCRIPTION

The present invention provides, among other things, methods andcompositions for delivering mRNA in vivo using improved liposomesincorporating a sterol-based cationic lipid, a helper lipid, and a PEGor PEG-modified lipid as described herein.

Lipid Nanoparticles

According to the present invention, suitable compositions describedherein comprise nucleic acids; and lipid nanoparticles encapsulating thenucleic acids, wherein said lipid nanoparticles comprise distinct lipidcomponents. In some embodiments, there are no more than three distinctlipid components and exemplary distinct lipid components includesterol-based cationic lipids, helper lipids (e.g., non-cationic lipids),and PEG-modified lipids.

In some embodiments, the lipid nanoparticles have an encapsulationpercentage of nucleic acids that is at least about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95%.

In some embodiments, the percentage of lipid nanoparticles in acomposition that encapsulate a nucleic acid is at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, or about 95%.

Sterol-Based Cationic Lipids

As used herein, the phrase “sterol-based lipid” or “sterol-basedcationic lipid” refers to a cationic lipid comprising a steroid moiety.As used herein, the phrase “cationic lipid” refers to any of a number oflipid species that carry a net positive charge at a selected pH, such asphysiological pH.

In embodiments, a sterol-based cationic lipid has a structure accordingto Formula (A),B-L¹-S  (Formula A),or a protonated form thereof.

B is a basic functional group (e.g., wherein the protonated form has apK_(a) that is no more than about 8.0).

L¹ is an optionally substituted linker group (e.g., a C₁-C₂₀ alkylene ora 2- to 20-membered heteroalkylene).

S is a sterol.

In some embodiments, B is a basic functional group wherein theprotonated form has a pK_(a) that is no more than about 9.0, about 8.5,about 8.0, about 7.5, or about 7.0. In embodiments, B is a basicfunctional group wherein the protonated form has a pK_(a) that is nomore than about 8.0 or about 7.5.

In some embodiments, B is a basic functional group wherein theprotonated form has a pK_(a) that is about 4.0 to about 9.0, about 4.0to about 8.0, about 4.5 to about 8.0, about 5.0 to about 8.0, about 5.5to about 8.0, or about 6.0 to about 8.0.

In some embodiments, B is an optionally substituted 5- or 6-memberednitrogen-containing heteroaryl.

In some embodiments, B is a group selected from pyrrolyl, imidazolyl,pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyrazinyl, andpyridazinyl, each of which is optionally substituted. For any optionallysubstituted group described herein (e.g., a group that has 0, 1, 2, 3,or 4 independent substituent groups), exemplary substituent groupsinclude but are not limited to: halogen (e.g., —F, —Cl, —Br, or —I),C₁₋₂₀ alkyl (e.g., a C₁₋₆ alkyl), C₁₋₂₀ haloalkyl (e.g., a C₁₋₆haloalkyl), —CN, —OH, —O(C₁₋₂₀ alkyl) (e.g., —O(C₁₋₆ alkyl)),—O(C₁₋₂₀haloalkyl) (e.g., —O(C₁₋₆ haloalkyl)), —NH₂, —NH(C₁₋₆ alkyl),and —NH(C₁₋₆ alkyl)₂.

In some embodiments, B is a group selected from pyrrolyl, pyrazolyl,triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyrazinyl, and pyridazinyl,each of which is optionally substituted.

In some embodiments, B is a group selected from pyrrolyl, pyrazolyl,triazolyl, tetrazolyl, pyrimidyl, pyrazinyl, and pyridazinyl, each ofwhich is optionally substituted.

In some embodiments, L¹ is a linker group that is optionally substitutedC₁-C₂₀ alkylene (e.g., an optionally substituted C₁-C₁₀ alkylene or anoptionally substituted C₁-C₅ alkylene). As used herein, the term“alkylene” represents a saturated divalent straight or branched chainhydrocarbon group and is exemplified by methylene, ethylene,isopropylene and the like.

In some embodiments, L¹ is a linker group that is optionally substituted2- to 20-membered heteroalkylene (e.g., an optionally substituted 2- to12-membered heteroalkylene or an optionally substituted 2- to 6-memberedheteroalkylene). As used herein, the term “heteroalkylene” refers to adivalent heteroalkyl group. The term “heteroalkyl group” refers to astable straight or branched chain hydrocarbon radical, having the statednumber of atoms, wherein the atoms are selected from carbon and from oneto three heteroatoms selected from the group consisting of O, N, Si andS, and wherein the nitrogen and sulfur atoms can optionally be oxidizedand the nitrogen heteroatom can optionally be quaternized. Theheteroatom(s) O, N and S can be placed at any interior position of theheteroalkyl group.

In some embodiments, L¹ is a linker group that is optionally substituted2- to 20-membered heteroalkylene, and wherein L¹ is non-peptidic (thatis, a non-peptidic linker L¹ is one that does not comprise amino acidresidues).

In some embodiments, L¹ is a linker group that comprises a moiety thatis an ester group, an amide group, a carbamate group, a carbonate group,or a urea group.

In some embodiments, L¹ is a linker group that comprises a moiety thatis an amide group, a carbamate group, a carbonate group, or a ureagroup.

In some embodiments, L¹ is a linker group that comprises a moiety thatis an amide group, a carbonate group, or a urea group.

In some embodiments, L¹ is a linker group represented by a formula thatis: —X¹—C(X³)—X², —(C₁-C₁₉ alkylene)-X¹—C(X³)—X², —X¹—C(X³)—X²(C₁-C₁₉alkylene)-, —(C₁-C₁₉ alkylene)-X¹—, —X¹—(C₁-C₁₉ alkylene)-.

Each X¹ and X² is independently, a covalent bond, —O—, —S—, or —NH—.

X³ is independently ═O, ═S, or ═NH.

The C₁-C₁₉ alkylene group (e.g., a C₁-C₅ alkylene or a C₁-C₁₀ alkylenegroup) is independently optionally substituted.

In some embodiments, L¹ is unsubstituted.

In some embodiments, L¹ does not comprise substituents having thestructure —N(R′)₂, or a positively charged form thereof, wherein each R′is independently hydrogen or optionally substituted C₁-C₂₀ alkyl.

In some embodiments, S is a zoosterol, or an oxidized or reduced formthereof. In embodiments, S is a zoosterol. In embodiments, S is anoxidized form of a zoosterol. In embodiments, S is a reduced form of azoosterol.

In some embodiments, S is a phytosterol, or an oxidized or reduced formthereof. In embodiments, S is a phytosterol. In embodiments, S is anoxidized form of a phytosterol. In embodiments, S is a reduced form of aphytosterol.

In some embodiments, S is a synthetic sterol (e.g., non-naturallyoccurring), or an oxidized or reduced form thereof. In embodiments, S isa synthetic sterol. In embodiments, S is an oxidized form of a syntheticsterol. In embodiments, S is a reduced form of a synthetic sterol.

In some embodiments, S is an oxidized form of a sterol as describedherein.

In some embodiments, an oxidized form of a sterol is one in which theparent sterol has been modified to include further oxygen-containinggroups. In embodiments, an oxidized form of a sterol includes one ormore (e.g., 1, 2, 3, or 4) additional hydroxyl groups and/orcarbonyl-containing groups (e.g., a ketone, an aldehyde, a carboxylicacid, or a carboxylic ester moiety) as compared to the parent sterol.

In some embodiments, an oxidized form of a sterol is one in which theparent sterol has been modified to include unsaturated carbon-carbonbonds (e.g., carbon-carbon double bonds). In embodiments, an oxidizedform of a sterol includes one or more (e.g., 1, 2, or 3) additionalcarbon-carbon double bonds as compared to the parent sterol.

In some embodiments, S is a reduced form of a sterol as describedherein.

In some embodiments, a reduced form of a sterol is one in which theparent sterol has been modified to include fewer oxygen-containinggroups. In embodiments, a reduced form of a sterol includes a reducednumber (e.g., 1, 2, 3, or 4 fewer moieties) of hydroxyl groups and/orcarbonyl-containing groups (e.g., a ketone, an aldehyde, a carboxylicacid, or a carboxylic ester moiety) as compared to the parent sterol.

In some embodiments, a reduced form of a sterol is one in which theparent sterol has been modified to include fewer unsaturatedcarbon-carbon bonds (e.g., carbon-carbon double bonds). In embodiments,a reduced form of a sterol includes a reduced number (e.g., 1, 2, or 3fewer) of carbon-carbon double bonds as compared to the parent sterol.

In some embodiments, S is a sterol that is cholesterol, alkyllithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, orsitosterol, or any oxidized or reduced form thereof.

In some embodiments, S is a sterol selected from cholesterol, anoxidized form of cholesterol, a reduced form of cholesterol, alkyllithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, andsitosterol.

In some embodiments, S is a sterol selected from an oxidized form ofcholesterol, a reduced form of cholesterol, alkyl lithocholate,stigmasterol, stigmastanol, campesterol, ergosterol, and sitosterol.

In some embodiments, S is a sterol that is cholesterol, alkyllithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, orsitosterol.

In some embodiments, S is a sterol that is an oxidized form of:cholesterol, alkyl lithocholate, stigmasterol, stigmastanol,campesterol, ergosterol, or sitosterol.

In some embodiments, S is a sterol that is a reduced form of:cholesterol, alkyl lithocholate, stigmasterol, stigmastanol,campesterol, ergosterol, or sitosterol.

In some embodiments, S is a sterol that is cholesterol, an oxidized formof cholesterol, or a reduced form of cholesterol.

In some embodiments, S is a sterol that is alkyl lithocholate, anoxidized form of alkyl lithocholate, or a reduced form of alkyllithocholate.

In some embodiments, S is a sterol that is stigmasterol, an oxidizedform of stigmasterol, or a reduced form of stigmasterol.

In some embodiments, S is a sterol that is stigmastanol, an oxidizedform of stigmastanol, or a reduced form of stigmastanol.

In some embodiments, S is a sterol that is campesterol, an oxidized formof campesterol, or a reduced form of campesterol.

In some embodiments, S is a sterol that is ergosterol, an oxidized formof ergosterol, or a reduced form of ergosterol.

In some embodiments, S is a sterol that is sitosterol, an oxidized formof sitosterol, or a reduced form of sitosterol.

In some embodiments, S is a sterol that is a bile acid or an alkyl esterthereof, or an oxidized form thereof, or a reduced form thereof.

In some embodiments, S is a sterol that is cholic acid or an alkyl esterthereof, or an oxidized form thereof, or a reduced form thereof.

In some embodiments, S is a sterol selected from

wherein R is optionally substituted C₁-C₂₀ alkyl, and R′ is H oroptionally substituted C₁-C₂₀ alkyl.

In some embodiments, S is a sterol that is

In some embodiments, S is a sterol selected from

wherein R is optionally substituted C₁-C₂₀ alkyl, and R′ is H oroptionally substituted C₁-C₂₀ alkyl.

In some embodiments, a sterol-based cationic lipid comprises imidazolecholesterol ester (ICE).

In some embodiments, a sterol-based cationic lipid does not compriseimidazole cholesterol ester (ICE).

In some embodiments, a sterol-based cationic lipid is acholesterol-based cationic lipid. Such cholesterol-based cationic lipidscan be used, either alone or in combination with other lipids asdescribed herein. Suitable cholesterol-based cationic lipids include,for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335).

In embodiments, sterol-based cationic lipids are dialkylamino-,imidazole-, and guanidinium-containing sterol-based cationic lipids. Forexample, certain embodiments are directed to a composition comprisingone or more sterol-based cationic lipids comprising an imidazole, forexample, the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R,17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10,11, 12, 13, 14, 15, 16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I) below.In certain embodiments, a lipid nanoparticle for delivery of RNA (e.g.,mRNA) encoding a functional protein may comprise one or moreimidazole-based cationic lipids, for example, the imidazole cholesterolester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I).

An exemplary hydrolysis reaction of structure (I) is provided in Scheme1.

Without wishing to be bound by a particular theory, it is believed thatthe fusogenicity of the imidazole-based cationic lipid ICE is related tothe endosomal disruption which is facilitated by the imidazole group,which has a lower pKa relative to traditional cationic lipids. Theendosomal disruption in turn promotes osmotic swelling and thedisruption of the liposomal membrane, followed by the transfection orintracellular release of the polynucleotide contents loaded orencapsulated therein into the target cell.

The imidazole-based cationic lipids are also characterized by theirreduced toxicity relative to other cationic lipids. In some embodiments,one or more of the lipid nanoparticles which comprises the blendedpharmaceutical composition comprise an imidazole-based cationic lipidsuch as ICE, to reduce the relative concentration of other more toxiccationic lipids in such blended pharmaceutical composition. Theimidazole-based cationic lipids (e.g., ICE) may be used as the solecationic lipid in one or more of the lipid nanoparticles that comprisethe blended formulations, or alternatively may be combined withtraditional cationic lipids (e.g., DOPE, DC-Chol), non-cationic lipids,PEG-modified lipids and/or helper lipids. The cationic lipid maycomprise a molar ratio of about 1% to about 90%, about 2% to about 90%,about 5% to about 90%, about 10% to about 90%, about 15% to about 90%,about 20% to about 90%, about 30% to about 90%, about 40% to about 90%,about 50% to about 90%, about 60% to about 90%, about 70% to about 90%,about 80% to about 90%, about 1% to about 80%, about 1% to about 70%,about 1% to about 60%, about 1% to about 50%, about 1% to about 40%,about 1% to about 30%, about 1% to about 20%, about 1% to about 15%,about 1% to about 10%, about 2% to about 70%, about 5% to about 50%,about 10% to about 40% of the total lipid present in the lipidnanoparticle, or preferably about 20% to about 70% of the total lipidpresent in the lipid nanoparticle.

Ratio of Distinct Lipid Components

In embodiments where a lipid nanoparticle comprises three and no morethan three distinct lipid components, the ratio of total lipid content(i.e., the ratio of lipid component (1):lipid component (2):lipidcomponent (3)) can be represented as x:y:z, wherein(y+z)=100−x.

In some embodiments, each of “x,” “y,” and “z” represents molarpercentages of the three distinct lipid components, and the ratio is amolar ratio.

In some embodiments, each of “x,” “y,” and “z” represents weightpercentages of the three distinct lipid components, and the ratio is aweight ratio.

In some embodiments, lipid component (1), represented by variable “x,”is a sterol-based cationic lipid.

In some embodiments, lipid component (2), represented by variable “y,”is a helper lipid.

In some embodiments, lipid component (3), represented by variable “z”,is a PEG lipid.

In some embodiments, variable “x,” representing the molar percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95%.

In some embodiments, variable “x,” representing the molar percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is no morethan about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about20%, or about 10%. In some embodiments, variable “x” is no more thanabout 65%, about 60%, about 55%, about 50%, or about 40%.

In some embodiments, variable “x,” representing the molar percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is: at leastabout 50% but less than about 95%; at least about 50% but less thanabout 90%; at least about 50% but less than about 85%; at least about50% but less than about 80%; at least about 50% but less than about 75%;at least about 50% but less than about 70%; at least about 50% but lessthan about 65%; or at least about 50% but less than about 60%.

In some embodiments, variable “x,” representing the weight percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95%.

In some embodiments, variable “x,” representing the weight percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is no morethan about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about20%, or about 10%.

In some embodiments, variable “x,” representing the weight percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is: at leastabout 50% but less than about 95%; at least about 50% but less thanabout 90%; at least about 50% but less than about 85%; at least about50% but less than about 80%; at least about 50% but less than about 75%;at least about 50% but less than about 70%; at least about 50% but lessthan about 65%; or at least about 50% but less than about 60%.

In some embodiments, variable “z,” representing the molar percentage oflipid component (3) (e.g., a PEG-modified lipid) is no more than about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In someembodiments, variable “z,” representing the molar percentage of lipidcomponent (3) (e.g., a PEG-modified lipid) is about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%. In embodiments, variable “z,” representing themolar percentage of lipid component (3) (e.g., a PEG-modified lipid) isabout 1% to about 10%, about 2% to about 10%, about 3% to about 10%,about 4% to about 10%, about 5% to about 10%, about 6% to about 10%,about 7% to about 10%, about 8% to about 10%, about 9% to about 10%,about 1% to about 9%, about 1% to about 8%, about 1% to about 7.5%,about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2.5%to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5%to about 7.5%, or about 5% to about 10%.

In some embodiments, variable “z,” representing the weight percentage oflipid component (3) (e.g., a PEG-modified lipid) is no more than about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In someembodiments, variable “z,” representing the weight percentage of lipidcomponent (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%. In some embodiments, variable “z,” representing the weightpercentage of lipid component (3) (e.g., a PEG-modified lipid) is about1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4%to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% toabout 10%, about 8% to about 10%, about 9% to about 10%, about 1% toabout 9%, about 1% to about 8%, about 1% to about 7.5%, about 1% toabout 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about4%, about 1% to about 3%, about 1% to about 2%, about 2.5% to about 10%,about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about7.5%, or about 5% to about 10%.

For compositions having three and only three distinct lipid components,variables “x,” “y,” and “z” may be in any combination so long as thetotal of the three variables sums to 100% of the total lipid content.

Other Lipid Components

The use of polyethylene glycol (PEG)-modified phospholipids andderivatized lipids such as derivatized cerarmides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention,either alone or preferably in combination with other lipid formulationstogether which comprise the transfer vehicle (e.g., a lipidnanoparticle). Contemplated PEG-modified lipids include, but are notlimited to, a polyethylene glycol chain of up to 5 kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C6-C20 length. Theaddition of such components may prevent complex aggregation and may alsoprovide a means for increasing circulation lifetime and increasing thedelivery of the lipid-nucleic acid composition to the target tissues,(Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may beselected to rapidly exchange out of the formulation in vivo (see U.S.Pat. No. 5,885,613). Particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C14 or C18).

The PEG-modified phospholipid and derivitized lipids of the presentinvention may comprise a molar ratio from about 0% to about 20%, about0.5% to about 20%, about 1% to about 20%, about 2% to about 20%, about3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 10%to about 20%, about 15% to about 20%, about 0% to about 15%, about 0% toabout 10%, about 0% to about 5%, about 0% to about 4%, about 0% to about3%, about 0% to about 220%, about 0% to about 1%, about 5% to about 15%,about 4% to about 10%, or about 2% of the total lipid present in theliposomal transfer vehicle.

The present invention also contemplates the use of non-cationic lipids.As used herein, the phrase “non-cationic lipid” refers to any neutral,zwitterionic or anionic lipid. As used herein, the phrase “anioniclipid” refers to any of a number of lipid species that carry a netnegative charge at a selected pH, such as physiological pH. Non-cationiclipids include, but are not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. Such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids.

In some embodiments, cationic lipids that are not sterol-based cationiclipids can be used in liposomal compositions of the invention. Cationiclipids include, but are not limited to,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),5-carboxyspermylglycinedioctadecylamide (DOGS),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium(DOSPA), 1,2-Dioleoyl-3-Dimethylammonium-Propane (DODAP),1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA),N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane(CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′,1-2′-octadecadienoxy)propane (CpLinDMA),N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP),1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or (DLinCDAP),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA),or mixtures thereof.

Additional cationic lipids include2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine(further described in U.S. Provisional Application No:PCT/US2012/041663, filed Jun. 8, 2012, the entire teachings of which areincorporated herein by reference in their entirety), cleavable lipids,such as, for example, one or more cationic lipids that comprise acleavable disulfide (S—S) functional group, as further described in U.S.International Application No: PCT/US2012/041663. In addition, severalreagents are commercially available to enhance transfection efficacy.Suitable examples include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad,Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000(Invitrogen), FUGENE, TRANSFECTAM (DOGS), and EFFECTENE.

Formation of Liposomes Encapsulating mRNA

The liposomal transfer vehicles for use in the present invention can beprepared by various techniques which are presently known in the art.Multi-lamellar vesicles (MLV) may be prepared conventional techniques,for example, by depositing a selected lipid on the inside wall of asuitable container or vessel by dissolving the lipid in an appropriatesolvent, and then evaporating the solvent to leave a thin film on theinside of the vessel or by spray drying. An aqueous phase may then beadded to the vessel with a vortexing motion which results in theformation of MLVs. Uni-lamellar vesicles (ULV) can then be formed byhomogenization, sonication or extrusion of the multi-lamellar vesicles.In addition, unilamellar vesicles can be formed by detergent removaltechniques.

In certain embodiments of this invention, the compositions of thepresent invention comprise a transfer vehicle wherein the therapeuticRNA (e.g., mRNA encoding CFTR) is associated on both the surface of thetransfer vehicle (e.g., a liposome) and encapsulated within the sametransfer vehicle. For example, during preparation of the compositions ofthe present invention, cationic liposomal transfer vehicles mayassociate with the nucleic acids (e.g., mRNA) through electrostaticinteractions with such therapeutic mRNA.

In some embodiments, the compositions and methods of the inventioncomprise mRNA encapsulated in a liposome. In some embodiments, the oneor more mRNA species may be encapsulated in the same liposome. In someembodiments, the one or more mRNA species may be encapsulated indifferent liposomes. In some embodiments, the mRNA is encapsulated inone or more liposomes, which differ in their lipid composition, molarratio of lipid components, size, charge (Zeta potential), targetingligands and/or combinations thereof. In some embodiments, the one ormore liposome may have a different composition of sterol-based cationiclipids, neutral lipid, PEG-modified lipid and/or combinations thereof.In some embodiments the one or more liposomes may have a different molarratio of sterol-based cationic lipid, neutral lipid, and PEG-modifiedlipid used to create the liposome.

The process of incorporation of a desired mRNA into a liposome is oftenreferred to as “loading”. Exemplary methods are described in Lasic, etal., FEBS Lett., 312: 255-258, 1992, which is incorporated herein byreference. The liposome-incorporated nucleic acids may be completely orpartially located in the interior space of the liposome, within thebilayer membrane of the liposome, or associated with the exteriorsurface of the liposome membrane. The incorporation of a nucleic acidinto liposomes is also referred to herein as “encapsulation” wherein thenucleic acid is entirely contained within the interior space of theliposome. The purpose of incorporating an mRNA into a transfer vehicle,such as a liposome, is often to protect the nucleic acid from anenvironment which may contain enzymes or chemicals that degrade nucleicacids and/or systems or receptors that cause the rapid excretion of thenucleic acids. Accordingly, in some embodiments, a suitable deliveryvehicle is capable of enhancing the stability of the mRNA containedtherein and/or facilitate the delivery of mRNA to the target cell ortissue.

Any desired lipids may be mixed at any ratios suitable for encapsulatingmRNAs. In some embodiments, a suitable lipid solution contains a mixtureof desired lipids including sterol-based cationic lipids, non-cationiclipids, and/or PEG-modified lipids. In some embodiments, a suitablelipid solution contain a mixture of desired lipids including one or moresterol-based cationic lipids, one or more helper lipids (e.g. noncationic lipids) and one or more PEG-modified lipids.

Exemplary combinations of sterol-based cationic lipids, non-cationiclipids, and PEG-modified lipids are described in the Examples section.For example, a suitable lipid solution may contain ICE, DOPE andDMG-PEG2K. The selection of sterol-based cationic lipids, non-cationiclipids and/or PEG-modified lipids which comprise the lipid mixture aswell as the relative molar ratio of such lipids to each other, is basedupon the characteristics of the selected lipid(s) and the nature of theand the characteristics of the mRNA to be encapsulated. Additionalconsiderations include, for example, the saturation of the alkyl chain,as well as the size, charge, pH, pKa, fusogenicity and toxicity of theselected lipid(s). Thus the molar ratios may be adjusted accordingly.

In some embodiments, a process for encapsulating mRNA in lipidnanoparticles comprises mixing an mRNA solution and a lipid solution,wherein the mRNA solution and/or the lipid solution are heated to apre-determined temperature greater than ambient temperature prior tomixing to form lipid nanoparticles that encapsulate mRNA (see U.S.patent application Ser. No. 14/790,562 entitled “Encapsulation ofmessenger RNA”, filed Jul. 2, 2015 and its provisional U.S. patentapplication Ser. No. 62/020,163, filed Jul. 2, 2014, the disclosure ofwhich are hereby incorporated in their entirety).

In some embodiments, a process for encapsulating mRNA in lipidnanoparticles comprises combining pre-formed lipid nanoparticles withmRNA (see U.S. Provisional Application Ser. No. 62/420,413, filed Nov.10, 2016 and U.S. Provisional Application Ser. No. 62/580,155, filedNov. 1, 2017, the disclosures of which are hereby incorporated byreference). In some embodiments, combining pre-formed lipidnanoparticles with mRNA results in lipid nanoparticles that showimproved efficacy of intracellular delivery of the mRNA. In someembodiments, combining pre-formed lipid nanoparticles with mRNA resultsin very high encapsulation efficiencies of mRNA encapsulated in lipidnanoparticles (i.e., in the range of 90-95%). In some embodiments,combining pre-formed lipid nanoparticles with mRNA is achieved with pumpsystems which maintain the lipid/mRNA (N/P) ratio constant throughoutthe process and which also afford facile scale-up.

Suitable liposomes in accordance with the present invention may be madein various sizes. In some embodiments, provided liposomes may be madesmaller than previously known mRNA encapsulating liposomes. In someembodiments, decreased size of liposomes is associated with moreefficient delivery of mRNA. Selection of an appropriate liposome sizemay take into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made.

In some embodiments, an appropriate size of liposome is selected tofacilitate systemic distribution of a protein or a peptide encoded bythe mRNA. In some embodiments, it may be desirable to limit delivery ofthe mRNA to certain cells or tissues. For example, to targethepatocytes, a liposome may be sized such that its dimensions aresmaller than the fenestrations of the endothelial layer lining hepaticsinusoids in the liver; in such cases the liposome could readilypenetrate such endothelial fenestrations to reach the targethepatocytes.

Alternatively or additionally, a liposome may be sized such that thedimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,a liposome may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the liposome particle. In some embodiments, asuitable liposome has a size no greater than about 250 nm (e.g., nogreater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75nm, or 50 nm). In some embodiments, a suitable liposome has a sizeranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm).In some embodiments, a suitable liposome has a size ranging from about100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm,100-150 nm). In some embodiments, a suitable liposome has a size rangingfrom about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70nm, 10-60 nm, or 10-50 nm). In a particular embodiment, a suitableliposome has a size less than about 100 nm. In some embodiments,majority of nanoparticles in a composition, i.e., greater than about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%of the purified nanoparticles, have a size less than about 100 nm (e.g.,less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm,about 45 nm, or about 40 nm). In some embodiments, substantially all ofthe purified nanoparticles have a size less than 100 nm (e.g., less thanabout 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm, about 45 nm,or about 40 nm).

A variety of alternative methods known in the art are available forsizing of a population of liposomes. One such sizing method is describedin U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicatinga liposome suspension either by bath or probe sonication produces aprogressive size reduction down to small ULV less than about 0.05microns in diameter. Homogenization is another method that relies onshearing energy to fragment large liposomes into smaller ones. In atypical homogenization procedure, MLV are recirculated through astandard emulsion homogenizer until selected liposome sizes, typicallybetween about 0.1 and 0.5 microns, are observed. The size of theliposomes may be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981),incorporated herein by reference. Average liposome diameter may bereduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Purification

Typically, subsequent to formulation the lipid nanoparticles containingmRNA are purified and/or concentrated. Various purification methods maybe used. In some embodiments, lipid nanoparticles are purified usingTangential Flow Filtration. Tangential flow filtration (TFF), alsoreferred to as cross-flow filtration, is a type of filtration whereinthe material to be filtered is passed tangentially across a filterrather than through it. In TFF, undesired permeate passes through thefilter, while the desired retentate passes along the filter and iscollected downstream. It is important to note that the desired materialis typically contained in the retentate in TFF, which is the opposite ofwhat one normally encounters in traditional-dead end filtration.

Depending upon the material to be filtered, TFF is usually used foreither microfiltration or ultrafiltration. Microfiltration is typicallydefined as instances where the filter has a pore size of between 0.05 μmand 1.0 μm, inclusive, while ultrafiltration typically involves filterswith a pore size of less than 0.05 μm. Pore size also determines thenominal molecular weight limits (NMWL), also referred to as themolecular weight cut off (MWCO) for a particular filter, withmicrofiltration membranes typically having NMWLs of greater than 1,000kilodaltons (kDa) and ultrafiltration filters having NMWLs of between 1kDa and 1,000 kDa.

A principal advantage of tangential flow filtration is thatnon-permeable particles that may aggregate in and block the filter(sometimes referred to as “filter cake”) during traditional “dead-end”filtration are instead carried along the surface of the filter. Thisadvantage allows tangential flow filtration to be widely used inindustrial processes requiring continuous operation since down time issignificantly reduced because filters do not generally need to beremoved and cleaned.

Tangential flow filtration can be used for several purposes includingconcentration and diafiltration, among others. Concentration is aprocess whereby solvent is removed from a solution while solutemolecules are retained. In order to effectively concentrate a sample, amembrane having a NMWL or MWCO that is substantially lower than themolecular weight of the solute molecules to be retained is used.Generally, one of skill may select a filter having a NMWL or MWCO ofthree to six times below the molecular weight of the target molecule(s).

Diafiltration is a fractionation process whereby small undesiredparticles are passed through a filter while larger desired nanoparticlesare maintained in the retentate without changing the concentration ofthose nanoparticles in solution. Diafiltration is often used to removesalts or reaction buffers from a solution. Diafiltration may be eithercontinuous or discontinuous. In continuous diafiltration, adiafiltration solution is added to the sample feed at the same rate thatfiltrate is generated. In discontinuous diafiltration, the solution isfirst diluted and then concentrated back to the starting concentration.Discontinuous diafiltration may be repeated until a desiredconcentration of nanoparticles is reached.

Purified and/or concentrated lipid nanoparticles may be formulated in adesired buffer such as, for example, PBS.

Thus, the present invention provides a composition comprising purifiednanoparticles described herein. In some embodiments, majority ofpurified nanoparticles in a composition, i.e., greater than about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ofthe purified nanoparticles, have a size less than about 100 nm (e.g.,less than about 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm,about 45 nm, or about 40 nm). In some embodiments, substantially all ofthe purified nanoparticles have a size less than 100 nm (e.g., less thanabout 95 nm, about 90 nm, about 85 nm, about 80 nm, about 75 nm, about70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm, about 45 nm,or about 40 nm).

In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% of the purified nanoparticles in a compositionprovided by the present invention have a size ranging from about 40-90nm (e.g., about 40-85 nm, about 40-80 nm, about 40-75 nm, about 40-70nm, about 40-65 nm, or about 40-60 nm). In some embodiments,substantially all of the purified nanoparticles have a size ranging fromabout 40-90 nm (e.g., about 40-85 nm, about 40-80 nm, about 40-75 nm,about 40-70 nm, about 40-65 nm, or about 40-60 nm).

In some embodiments, the dispersity, or measure of heterogeneity in sizeof molecules (PDI), of nanoparticles in a composition provided by thepresent invention is less than about 0.16 (e.g., less than about 0.15,0.14, 0.13, 0.12, 0.11, 0.10, 0.09, or 0.08).

In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% of the purified lipid nanoparticles in a compositionprovided by the present invention encapsulate an mRNA within eachindividual particle. In some embodiments, substantially all of thepurified lipid nanoparticles in a composition encapsulate an mRNA withineach individual particle.

In some embodiments, a composition according to the present inventioncontains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg ofencapsulated mRNA. In some embodiments, a process according to thepresent invention results in greater than about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

Nucleic Acids and mRNA

The present invention can be used to deliver any type of nucleic acid. Anucleic acid may be selected from the group comprising (but not limitedto) DNA, messenger RNA (mRNA), small nuclear RNA (snRNA), guide RNA(gRNA), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), micro RNA(miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA).

The present invention can be used to deliver any mRNA. mRNA is typicallythought of as the type of RNA that carries information from DNA to theribosome. Typically, in eukaryotic organisms, mRNA processing comprisesthe addition of a “cap” on the N-terminal (5′) end, and a “tail” on theC-terminal (3′) end. A typical cap is a 7-methylguanosine cap, which isa guanosine that is linked through a 5′-5′-triphosphate bond to thefirst transcribed nucleotide. The presence of the cap is important inproviding resistance to nucleases found in most eukaryotic cells. Thetail is typically a polyadenylation event whereby a polyadenylyl moietyis added to the 3′ end of the mRNA molecule. The presence of this “tail”serves to protect the mRNA from exonuclease degradation. Messenger RNAtypically is translated by the ribosomes into a series of amino acidsthat make up a protein or a peptide.

Any mRNA capable of being translated into one or more peptides (e.g.,antigens) or peptide fragments is contemplated as within the scope ofthe present invention. In some embodiments, an mRNA encodes one or morenaturally occurring peptides. In some embodiments, an mRNA encodes oneor more modified or non-natural peptides.

In some embodiments an mRNA encodes an intracellular protein or peptide.In some embodiments, an mRNA encodes a cytosolic protein or peptide. Insome embodiments, an mRNA encodes a protein or peptide associated withthe actin cytoskeleton. In some embodiments, an mRNA encodes a proteinor peptide associated with the plasma membrane. In some specificembodiments, an mRNA encodes a transmembrane protein or peptide. In somespecific embodiments an mRNA encodes an ion channel protein or peptide.In some embodiments, an mRNA encodes a perinuclear protein or peptide.In some embodiments, an mRNA encodes a nuclear protein or peptide. Insome specific embodiments, an mRNA encodes a transcription factor. Insome embodiments, an mRNA encodes a chaperone protein or peptide. Insome embodiments, an mRNA encodes an intracellular enzyme (e.g., mRNAencoding an enzyme associated with urea cycle or lysosomal storagemetabolic disorders). In some embodiments, an mRNA encodes a protein orpeptide involved in cellular metabolism, DNA repair, transcriptionand/or translation. In some embodiments, an mRNA encodes anextracellular protein or peptide. In some embodiments, an mRNA encodes aprotein or peptide associated with the extracellular matrix. In someembodiments an mRNA encodes a secreted protein or peptide. In specificembodiments, an mRNA used in the composition and methods of theinvention may be used to express functional proteins or enzymes that areexcreted or secreted by one or more target cells into the surroundingextracellular fluid (e.g., mRNA encoding hormones and/orneurotransmitters).

In some embodiments, the compositions and methods of the inventionprovide for delivery of mRNA encoding a secreted protein. In someembodiments, the compositions and methods of the invention provide fordelivery of mRNA encoding one or more secreted proteins listed in Table1; thus, compositions of the invention may comprise an mRNA encoding aprotein listed in Table 1 (or a homolog thereof) along with othercomponents set out herein, and methods of the invention may comprisepreparing and/or administering a composition comprising an mRNA encodinga protein listed in Table 1 (or a homolog thereof) along with othercomponents set out herein.

TABLE 1 Secreted Proteins Uniprot ID Protein Name Gene Name A1E959Odontogenic ameloblast-associated protein ODAM A1KZ92 Peroxidasin-likeprotein PXDNL A1L453 Serine protease 38 PRSS38 A1L4H1 Soluble scavengerreceptor cysteine-rich domain-containing protein SSC5D SSC5D A2RUU4Colipase-like protein 1 CLPSL1 A2VDF0 Fucose mutarotase FUOM A2VEC9SCO-spondin SSPO A3KMH1 von Willebrand factor A domain-containingprotein 8 VWA8 A4D0S4 Laminin subunit beta-4 LAMB4 A4D1T9 Probableinactive serine protease 37 PRSS37 A5D8T8 C-type lectin domain family 18member A CLEC18A A6NC86 phospholipase A2 inhibitor and Ly6/PLAURdomain-containing PINLYP protein A6NCI4 von Willebrand factor Adomain-containing protein 3A VWA3A A6ND01 Probable folate receptor deltaFOLR4 A6NDD2 Beta-defensin 108B-like A6NE02 BTB/POZ domain-containingprotein 17 BTBD17 A6NEF6 Growth hormone 1 GH1 A6NF02 NPIP-like proteinLOC730153 A6NFB4 HCG1749481, isoform CRA_k CSH1 A6NFZ4 Protein FAM24AFAM24A A6NG13 Glycosyltransferase 54 domain-containing protein A6NGN9IgLON family member 5 IGLON5 A6NHN0 Otolin-1 OTOL1 A6NHN6 Nuclear porecomplex-interacting protein-like 2 NPIPL2 A6NI73 Leukocyteimmunoglobulin-like receptor subfamily A member 5 LILRA5 A6NIT4Chorionic somatomammotropin hormone 2 isoform 2 CSH2 A6NJ69 IgA-inducingprotein homolog IGIP A6NKQ9 Choriogonadotropin subunit beta variant 1CGB1 A6NMZ7 Collagen alpha-6(VI) chain COL6A6 A6NNS2Dehydrogenase/reductase SDR family member 7C DHRS7C A6XGL2 Insulin Achain INS A8K0G1 Protein Wnt WNT7B A8K2U0 Alpha-2-macroglobulin-likeprotein 1 A2ML1 A8K7I4 Calcium-activated chloride channel regulator 1CLCA1 A8MTL9 Serpin-like protein HMSD HMSD A8MV23 Serpin E3 SERPINE3A8MZH6 Oocyte-secreted protein 1 homolog OOSP1 A8TX70 Collagenalpha-5(VI) chain COL6A5 B0ZBE8 Natriuretic peptide NPPA B1A4G9Somatotropin GH1 B1A4H2 HCG1749481, isoform CRA_d CSH1 B1A4H9 Chorionicsomatomammotropin hormone CSH2 B1AJZ6 Protein Wnt WNT4 B1AKI9 Isthmin-1ISM1 B2RNN3 Complement C1q and tumor necrosis factor-related protein 9BC1QTNF9B B2RUY7 von Willebrand factor C domain-containing protein 2-likeVWC2L B3GLJ2 Prostate and testis expressed protein 3 PATE3 B4DI03SEC11-like 3 (S. cerevisiae), isoform CRA_a SEC11L3 B4DJF9 Protein WntWNT4 B4DUL4 SEC11-like 1 (S. cerevisiae), isoform CRA_d SEC11L1 B5MCC8Protein Wnt WNT10B B8A595 Protein Wnt WNT7B B8A597 Protein Wnt WNT7BB8A598 Protein Wnt WNT7B B9A064 Immunoglobulin lambda-like polypeptide 5IGLL5 C9J3H3 Protein Wnt WNT10B C9J8I8 Protein Wnt WNT5A C9JAF2Insulin-like growth factor II Ala-25 Del IGF2 C9JCI2 Protein Wnt WNT10BC9JL84 HERV-H LTR-associating protein 1 HHLA1 C9JNR5 Insulin A chain INSC9JUI2 Protein Wnt WNT2 D6RF47 Protein Wnt WNT8A D6RF94 Protein WntWNT8A E2RYF7 Protein PBMUCL2 HCG22 E5RFR1 PENK(114-133) PENK E7EML9Serine protease 44 PRSS44 E7EPC3 Protein Wnt WNT9B E7EVP0 NociceptinPNOC E9PD02 Insulin-like growth factor I IGF1 E9PH60 Protein Wnt WNT16E9PJL6 Protein Wnt WNT11 F5GYM2 Protein Wnt WNT5B F5H034 Protein WntWNT5B F5H364 Protein Wnt WNT5B F5H7Q6 Protein Wnt WNT5B F8WCM5 ProteinINS-IGF2 INS-IGF2 F8WDR1 Protein Wnt WNT2 H0Y663 Protein Wnt WNT4 H0YK72Signal peptidase complex catalytic subunit SEC11A SEC11A H0YK83 Signalpeptidase complex catalytic subunit SEC11A SEC11A H0YM39 Chorionicsomatomammotropin hormone CSH2 H0YMT7 Chorionic somatomammotropinhormone CSH1 H0YN17 Chorionic somatomammotropin hormone CSH2 H0YNA5Signal peptidase complex catalytic subunit SEC11A SEC11A H0YNG3 Signalpeptidase complex catalytic subunit SEC11A SEC11A H0YNX5 Signalpeptidase complex catalytic subunit SEC11A SEC11A H7BZB8 Protein WntWNT10A H9KV56 Choriogonadotropin subunit beta variant 2 CGB2 I3L0L8Protein Wnt WNT9B J3KNZ1 Choriogonadotropin subunit beta variant 1 CGB1J3KP00 Choriogonadotropin subunit beta CGB7 J3QT02 Choriogonadotropinsubunit beta variant 1 CGB1 O00175 C—C motif chemokine 24 CCL24 O00182Galectin-9 LGALS9 O00187 Mannan-binding lectin serine protease 2 MASP2O00230 Cortistatin CORT O00253 Agouti-related protein AGRP O0027012-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptor GPR31 O00292Left-right determination factor 2 LEFTY2 O00294 Tubby-related protein 1TULP1 O00295 Tubby-related protein 2 TULP2 O00300 Tumor necrosis factorreceptor superfamily member 11B TNFRSF11B O00339 Matrilin-2 MATN2 O00391Sulfhydryl oxidase 1 QSOX1 O00468 Agrin AGRN O00515 Ladinin-1 LAD1O00533 Processed neural cell adhesion molecule L1-like protein CHL1O00584 Ribonuclease T2 RNASET2 O00585 C—C motif chemokine 21 CCL21O00602 Ficolin-1 FCN1 O00622 Protein CYR61 CYR61 O00626 MDC(5-69) CCL22O00634 Netrin-3 NTN3 O00744 Protein Wnt-10b WNT10B O00755 Protein Wnt-7aWNT7A O14498 Immunoglobulin superfamily containing leucine-rich repeatprotein ISLR O14511 Pro-neuregulin-2, membrane-bound isoform NRG2 O14594Neurocan core protein NCAN O14625 C—X—C motif chemokine 11 CXCL11 O14638Ectonucleotide pyrophosphatase/phosphodiesterase family member 3 ENPP3O14656 Torsin-1A TOR1A O14657 Torsin-1B TOR1B O14786 Neuropilin-1 NRP1O14788 Tumor necrosis factor ligand superfamily member 11, membraneTNFSF11 form O14791 Apolipoprotein L1 APOL1 O14793Growth/differentiation factor 8 MSTN O14904 Protein Wnt-9a WNT9A O14905Protein Wnt-9b WNT9B O14944 Proepiregulin EREG O14960 Leukocytecell-derived chemotaxin-2 LECT2 O15018 Processed PDZ domain-containingprotein 2 PDZD2 O15041 Semaphorin-3E SEMA3E O15072 A disintegrin andmetalloproteinase with thrombospondin motifs 3 ADAMTS3 O15123Angiopoietin-2 ANGPT2 O15130 Neuropeptide FF NPFF O15197 Ephrin type-Breceptor 6 EPHB6 O15204 ADAM DEC1 ADAMDEC1 O15230 Laminin subunitalpha-5 LAMA5 O15232 Matrilin-3 MATN3 O15240 Neuroendocrine regulatorypeptide-1 VGF O15263 Beta-defensin 4A DEFB4A O15335 Chondroadherin CHADO15393 Transmembrane protease serine 2 catalytic chain TMPRSS2 O15444C—C motif chemokine 25 CCL25 O15467 C—C motif chemokine 16 CCL16 O15496Group 10 secretory phospholipase A2 PLA2G10 O15520 Fibroblast growthfactor 10 FGF10 O15537 Retinoschisin RS1 O43157 Plexin-B1 PLXNB1 O43184Disintegrin and metalloproteinase domain-containing protein 12 ADAM12O43240 Kallikrein-10 KLK10 O43278 Kunitz-type protease inhibitor 1SPINT1 O43320 Fibroblast growth factor 16 FGF16 O43323 Desert hedgehogprotein C-product DHH O43405 Cochlin COCH O43508 Tumor necrosis factorligand superfamily member 12, membrane TNFSF12 form O43555Progonadoliberin-2 GNRH2 O43557 Tumor necrosis factor ligand superfamilymember 14, soluble form TNFSF14 O43692 Peptidase inhibitor 15 PI15O43699 Sialic acid-binding Ig-like lectin 6 SIGLEC6 O43820Hyaluronidase-3 HYAL3 O43827 Angiopoietin-related protein 7 ANGPTL7O43852 Calumenin CALU O43854 EGF-like repeat and discoidin I-likedomain-containing protein 3 EDIL3 O43866 CD5 antigen-like CD5L O43897Tolloid-like protein 1 TLL1 O43915 Vascular endothelial growth factor DFIGF O43927 C—X—C motif chemokine 13 CXCL13 O60218 Aldo-keto reductasefamily 1 member B10 AKR1B10 O60235 Transmembrane protease serine 11DTMPRSS11D O60258 Fibroblast growth factor 17 FGF17 O60259 Kallikrein-8KLK8 O60383 Growth/differentiation factor 9 GDF9 O60469 Down syndromecell adhesion molecule DSCAM O60542 Persephin PSPN O60565 Gremlin-1GREM1 O60575 Serine protease inhibitor Kazal-type 4 SPINK4 O60676Cystatin-8 CST8 O60687 Sushi repeat-containing protein SRPX2 SRPX2O60844 Zymogen granule membrane protein 16 ZG16 O60882 Matrixmetalloproteinase-20 MMP20 O60938 Keratocan KERA O75015 Low affinityimmunoglobulin gamma Fc region receptor III-B FCGR3B O75077 Disintegrinand metalloproteinase domain-containing protein 23 ADAM23 O75093 Slithomolog 1 protein SLIT1 O75094 Slit homolog 3 protein SLIT3 O75095Multiple epidermal growth factor-like domains protein 6 MEGF6 O75173 Adisintegrin and metalloproteinase with thrombospondin motifs 4 ADAMTS4O75200 Nuclear pore complex-interacting protein-like 1 NPIPL1 O75339Cartilage intermediate layer protein 1 C1 CILP O75354 Ectonucleosidetriphosphate diphosphohydrolase 6 ENTPD6 O75386 Tubby-related protein 3TULP3 O75398 Deformed epidermal autoregulatory factor 1 homolog DEAF1O75443 Alpha-tectorin TECTA O75445 Usherin USH2A O75462 Cytokinereceptor-like factor 1 CRLF1 O75487 Glypican-4 GPC4 O75493 Carbonicanhydrase-related protein 11 CA11 O75594 Peptidoglycan recognitionprotein 1 PGLYRP1 O75596 C-type lectin domain family 3 member A CLEC3AO75610 Left-right determination factor 1 LEFTY1 O75629 Protein CREG1CREG1 O75636 Ficolin-3 FCN3 O75711 Scrapie-responsive protein 1 SCRG1O75715 Epididymal secretory glutathione peroxidase GPX5 O75718Cartilage-associated protein CRTAP O75829 Chondrosurfactant proteinLECT1 O75830 Serpin I2 SERPINI2 O75882 Attractin ATRN O75888 Tumornecrosis factor ligand superfamily member 13 TNFSF13 O75900 Matrixmetalloproteinase-23 MMP23A O75951 Lysozyme-like protein 6 LYZL6 O75973C1q-related factor C1QL1 O76038 Secretagogin SCGN O76061 Stanniocalcin-2STC2 O76076 WNT1-inducible-signaling pathway protein 2 WISP2 O76093Fibroblast growth factor 18 FGF18 O76096 Cystatin-F CST7 O94769Extracellular matrix protein 2 ECM2 O94813 Slit homolog 2 proteinC-product SLIT2 O94907 Dickkopf-related protein 1 DKK1 O94919Endonuclease domain-containing 1 protein ENDOD1 O94964 N-terminal formSOGA1 O95025 Semaphorin-3D SEMA3D O95084 Serine protease 23 PRSS23O95150 Tumor necrosis factor ligand superfamily member 15 TNFSF15 O95156Neurexophilin-2 NXPH2 O95157 Neurexophilin-3 NXPH3 O95158Neurexophilin-4 NXPH4 O95388 WNT1-inducible-signaling pathway protein 1WISP1 O95389 WNT1-inducible-signaling pathway protein 3 WISP3 O95390Growth/differentiation factor 11 GDF11 O95393 Bone morphogenetic protein10 BMP10 O95399 Urotensin-2 UTS2 O95407 Tumor necrosis factor receptorsuperfamily member 6B TNFRSF6B O95428 Papilin PAPLN O95445Apolipoprotein M APOM O95450 A disintegrin and metalloproteinase withthrombospondin motifs 2 ADAMTS2 O95460 Matrilin-4 MATN4 O95467 LHALtetrapeptide GNAS O95631 Netrin-1 NTN1 O95633 Follistatin-relatedprotein 3 FSTL3 O95711 Lymphocyte antigen 86 LY86 O95715 C—X—C motifchemokine 14 CXCL14 O95750 Fibroblast growth factor 19 FGF19 O95760Interleukin-33 IL33 O95813 Cerberus CER1 O95841 Angiopoietin-relatedprotein 1 ANGPTL1 O95897 Noelin-2 OLFM2 O95925 Eppin EPPIN O95965Integrin beta-like protein 1 ITGBL1 O95967 EGF-containing fibulin-likeextracellular matrix protein 2 EFEMP2 O95968 Secretoglobin family 1Dmember 1 SCGB1D1 O95969 Secretoglobin family 1D member 2 SCGB1D2 O95970Leucine-rich glioma-inactivated protein 1 LGI1 O95972 Bone morphogeneticprotein 15 BMP15 O95994 Anterior gradient protein 2 homolog AGR2 O95998Interleukin-18-binding protein IL18BP O96009 Napsin-A NAPSA O96014Protein Wnt-11 WNT11 P00450 Ceruloplasmin CP P00451 Factor VIIIa lightchain F8 P00488 Coagulation factor XIII A chain F13A1 P00533 Epidermalgrowth factor receptor EGFR P00709 Alpha-lactalbumin LALBA P00734Prothrombin F2 P00738 Haptoglobin beta chain HP P00739Haptoglobin-related protein HPR P00740 Coagulation factor IXa heavychain F9 P00742 Factor X heavy chain F10 P00746 Complement factor D CFDP00747 Plasmin light chain B PLG P00748 Coagulation factor XIIa lightchain F12 P00749 Urokinase-type plasminogen activator long chain A PLAUP00750 Tissue-type plasminogen activator PLAT P00751 Complement factor BBa fragment CFB P00797 Renin REN P00973 2′-5′-oligoadenylate synthase 1OAS1 P00995 Pancreatic secretory trypsin inhibitor SPINK1 P01008Antithrombin-III SERPINC1 P01009 Alpha-1-antitrypsin SERPINA1 P01011Alpha-1-antichymotrypsin His-Pro-less SERPINA3 P01019 Angiotensin-1 AGTP01023 Alpha-2-macroglobulin A2M P01024 Acylation stimulating protein C3P01031 Complement C5 beta chain C5 P01033 Metalloproteinase inhibitor 1TIMP1 P01034 Cystatin-C CST3 P01036 Cystatin-S CST4 P01037 Cystatin-SNCST1 P01042 Kininogen-1 light chain KNG1 P01127 Platelet-derived growthfactor subunit B PDGFB P01135 Transforming growth factor alpha TGFAP01137 Transforming growth factor beta-1 TGFB1 P01138 Beta-nerve growthfactor NGF P01148 Gonadoliberin-1 GNRH1 P01160 Atrial natriuretic factorNPPA P01178 Oxytocin OXT P01185 Vasopressin-neurophysin 2-copeptin AVPP01189 Corticotropin POMC P01210 PENK(237-258) PENK P01213Alpha-neoendorphin PDYN P01215 Glycoprotein hormones alpha chain CGAP01222 Thyrotropin subunit beta TSHB P01225 Follitropin subunit betaFSHB P01229 Lutropin subunit beta LHB P01233 Choriogonadotropin subunitbeta CGB8 P01236 Prolactin PRL P01241 Somatotropin GH1 P01242 Growthhormone variant GH2 P01243 Chorionic somatomammotropin hormone CSH2P01258 Katacalcin CALCA P01266 Thyroglobulin TG P01270 Parathyroidhormone PTH P01275 Glucagon GCG P01282 Intestinal peptide PHM-27 VIPP01286 Somatoliberin GHRH P01298 Pancreatic prohormone PPY P01303C-flanking peptide of NPY NPY P01308 Insulin INS P01344 Insulin-likegrowth factor II IGF2 P01350 Big gastrin GAST P01374 Lymphotoxin-alphaLTA P01375 C-domain 1 TNF P01562 Interferon alpha-1/13 IFNA1 P01563Interferon alpha-2 IFNA2 P01566 Interferon alpha-10 IFNA10 P01567Interferon alpha-7 IFNA7 P01568 Interferon alpha-21 IFNA21 P01569Interferon alpha-5 IFNA5 P01570 Interferon alpha-14 IFNA14 P01571Interferon alpha-17 IFNA17 P01574 Interferon beta IFNB1 P01579Interferon gamma IFNG P01583 Interleukin-1 alpha IL1A P01584Interleukin-1 beta IL1B P01588 Erythropoietin EPO P01591 ImmunoglobulinJ chain IGJ P01732 T-cell surface glycoprotein CD8 alpha chain CD8AP01833 Polymeric immunoglobulin receptor PIGR P01857 Ig gamma-1 chain Cregion IGHG1 P01859 Ig gamma-2 chain C region IGHG2 P01860 Ig gamma-3chain C region IGHG3 P01861 Ig gamma-4 chain C region IGHG4 P01871 Ig muchain C region IGHM P01880 Ig delta chain C region IGHD P02452 Collagenalpha-1(I) chain COL1A1 P02458 Chondrocalcin COL2A1 P02461 Collagenalpha-1(III) chain COL3A1 P02462 Collagen alpha-1(IV) chain COL4A1P02647 Apolipoprotein A-I APOA1 P02649 Apolipoprotein E APOE P02652Apolipoprotein A-II APOA2 P02654 Apolipoprotein C-I APOC1 P02655Apolipoprotein C-II APOC2 P02656 Apolipoprotein C-III APOC3 P02671Fibrinogen alpha chain FGA P02675 Fibrinopeptide B FGB P02679 Fibrinogengamma chain FGG P02741 C-reactive protein CRP P02743 Serum amyloidP-component(1-203) APCS P02745 Complement C1q subcomponent subunit AC1QA P02746 Complement C1q subcomponent subunit B C1QB P02747 ComplementC1q subcomponent subunit C C1QC P02748 Complement component C9b C9P02749 Beta-2-glycoprotein 1 APOH P02750 Leucine-richalpha-2-glycoprotein LRG1 P02751 Ugl-Y2 FN1 P02753 Retinol-bindingprotein 4 RBP4 P02760 Trypstatin AMBP P02763 Alpha-1-acid glycoprotein 1ORM1 P02765 Alpha-2-HS-glycoprotein chain A AHSG P02766 TransthyretinTTR P02768 Serum albumin ALB P02771 Alpha-fetoprotein AFP P02774 VitaminD-binding protein GC P02775 Connective tissue-activating peptide IIIPPBP P02776 Platelet factor 4 PF4 P02778 CXCL10(1-73) CXCL10 P02786Transferrin receptor protein 1 TFRC P02787 Serotransferrin TF P02788Lactoferroxin-C LTF P02790 Hemopexin HPX P02808 Statherin STATH P02810Salivary acidic proline-rich phosphoprotein 1/2 PRH2 P02812 Basicsalivary proline-rich protein 2 PRB2 P02814 Peptide D1A SMR3B P02818Osteocalcin BGLAP P03950 Angiogenin ANG P03951 Coagulation factor XIaheavy chain F11 P03952 Plasma kallikrein KLKB1 P03956 27 kDainterstitial collagenase MMP1 P03971 Muellerian-inhibiting factor AMHP03973 Antileukoproteinase SLPI P04003 C4b-binding protein alpha chainC4BPA P04004 Somatomedin-B VTN P04054 Phospholipase A2 PLA2G1B P04085Platelet-derived growth factor subunit A PDGFA P04090 Relaxin A chainRLN2 P04114 Apolipoprotein B-100 APOB P04118 Colipase CLPS P04141Granulocyte-macrophage colony-stimulating factor CSF2 P04155 Trefoilfactor 1 TFF1 P04180 Phosphatidylcholine-sterol acyltransferase LCATP04196 Histidine-rich glycoprotein HRG P04217 Alpha-1B-glycoprotein A1BGP04275 von Willebrand antigen 2 VWF P04278 Sex hormone-binding globulinSHBG P04279 Alpha-inhibin-31 SEMG1 P04280 Basic salivary proline-richprotein 1 PRB1 P04628 Proto-oncogene Wnt-1 WNT1 P04745 Alpha-amylase 1AMY1A P04746 Pancreatic alpha-amylase AMY2A P04808 Prorelaxin H1 RLN1P05000 Interferon omega-1 IFNW1 P05013 Interferon alpha-6 IFNA6 P05014Interferon alpha-4 IFNA4 P05015 Interferon alpha-16 IFNA16 P05019Insulin-like growth factor I IGF1 P05060 GAWK peptide CHGB P05090Apolipoprotein D APOD P05109 Protein S100-A8 S100A8 P05111 Inhibin alphachain INHA P05112 Interleukin-4 IL4 P05113 Interleukin-5 IL5 P05120Plasminogen activator inhibitor 2 SERPINB2 P05121 Plasminogen activatorinhibitor 1 SERPINE1 P05154 Plasma serine protease inhibitor SERPINA5P05155 Plasma protease C1 inhibitor SERPING1 P05156 Complement factor Iheavy chain CFI P05160 Coagulation factor XIII B chain F13B P05161Ubiquitin-like protein ISG15 ISG15 P05230 Fibroblast growth factor 1FGF1 P05231 Interleukin-6 IL6 P05305 Big endothelin-1 EDN1 P05408C-terminal peptide SCG5 P05451 Lithostathine-1-alpha REG1A P05452Tetranectin CLEC3B P05543 Thyroxine-binding globulin SERPINA7 P05814Beta-casein CSN2 P05997 Collagen alpha-2(V) chain COL5A2 P06276Cholinesterase BCHE P06307 Cholecystokinin-12 CCK P06396 Gelsolin GSNP06681 Complement C2 C2 P06702 Protein S100-A9 S100A9 P06727Apolipoprotein A-IV APOA4 P06734 Low affinity immunoglobulin epsilon Fcreceptor soluble form FCER2 P06744 Glucose-6-phosphate isomerase GPIP06850 Corticoliberin CRH P06858 Lipoprotein lipase LPL P06881Calcitonin gene-related peptide 1 CALCA P07093 Glia-derived nexinSERPINE2 P07098 Gastric triacylglycerol lipase LIPF P07225 VitaminK-dependent protein S PROS1 P07237 Protein disulfide-isomerase P4HBP07288 Prostate-specific antigen KLK3 P07306 Asialoglycoprotein receptor1 ASGR1 P07355 Annexin A2 ANXA2 P07357 Complement component C8 alphachain C8A P07358 Complement component C8 beta chain C8B P07360Complement component C8 gamma chain C8G P07477 Alpha-trypsin chain 2PRSS1 P07478 Trypsin-2 PRSS2 P07492 Neuromedin-C GRP P07498 Kappa-caseinCSN3 P07585 Decorin DCN P07911 Uromodulin UMOD P07942 Laminin subunitbeta-1 LAMB1 P07988 Pulmonary surfactant-associated protein B SFTPBP07998 Ribonuclease pancreatic RNASE1 P08118 Beta-microseminoproteinMSMB P08123 Collagen alpha-2(I) chain COL1A2 P08185Corticosteroid-binding globulin SERPINA6 P08217 Chymotrypsin-likeelastase family member 2A CELA2A P08218 Chymotrypsin-like elastasefamily member 2B CELA2B P08253 72 kDa type IV collagenase MMP2 P08254Stromelysin-1 MMP3 P08294 Extracellular superoxide dismutase [Cu—Zn]SOD3 P08476 Inhibin beta A chain INHBA P08493 Matrix Gla protein MGPP08572 Collagen alpha-2(IV) chain COL4A2 P08581 Hepatocyte growth factorreceptor MET P08603 Complement factor H CFH P08620 Fibroblast growthfactor 4 FGF4 P08637 Low affinity immunoglobulin gamma Fc regionreceptor III-A FCGR3A P08697 Alpha-2-antiplasmin SERPINF2 P08700Interleukin-3 IL3 P08709 Coagulation factor VII F7 P08833 Insulin-likegrowth factor-binding protein 1 IGFBP1 P08887 Interleukin-6 receptorsubunit alpha IL6R P08949 Neuromedin-B-32 NMB P08F94 Fibrocystin PKHD1P09038 Fibroblast growth factor 2 FGF2 P09228 Cystatin-SA CST2 P09237Matrilysin MMP7 P09238 Stromelysin-2 MMP10 P09341 Growth-regulated alphaprotein CXCL1 P09382 Galectin-1 LGALS1 P09466 Glycodelin PAEP P09486SPARC SPARC P09529 Inhibin beta B chain INHBB P09544 Protein Wnt-2 WNT2P09603 Processed macrophage colony-stimulating factor 1 CSF1 P09681Gastric inhibitory polypeptide GIP P09683 Secretin SCT P09919Granulocyte colony-stimulating factor CSF3 P0C091 FRAS1-relatedextracellular matrix protein 3 FREM3 P0C0L4 C4d-A C4A P0C0L5 ComplementC4-B alpha chain C4B P0C0P6 Neuropeptide S NPS P0C7L1 Serine proteaseinhibitor Kazal-type 8 SPINK8 P0C862 Complement C1q and tumor necrosisfactor-related protein 9A C1QTNF9 P0C8F1 Prostate and testis expressedprotein 4 PATE4 P0CG01 Gastrokine-3 GKN3P P0CG36 Cryptic family protein1B CFC1B P0CG37 Cryptic protein CFC1 P0CJ68 Humanin-like protein 1MTRNR2L1 P0CJ69 Humanin-like protein 2 MTRNR2L2 P0CJ70 Humanin-likeprotein 3 MTRNR2L3 P0CJ71 Humanin-like protein 4 MTRNR2L4 P0CJ72Humanin-like protein 5 MTRNR2L5 P0CJ73 Humanin-like protein 6 MTRNR2L6P0CJ74 Humanin-like protein 7 MTRNR2L7 P0CJ75 Humanin-like protein 8MTRNR2L8 P0CJ76 Humanin-like protein 9 MTRNR2L9 P0CJ77 Humanin-likeprotein 10 MTRNR2L10 P0DJD7 Pepsin A-4 PGA4 P0DJD8 Pepsin A-3 PGA3P0DJD9 Pepsin A-5 PGA5 P0DJI8 Amyloid protein A SAA1 P0DJI9 Serumamyloid A-2 protein SAA2 P10082 Peptide YY(3-36) PYY P10092 Calcitoningene-related peptide 2 CALCB P10124 Serglycin SRGN P10145 MDNCF-a IL8P10147 MIP-1-alpha(4-69) CCL3 P10163 Peptide P-D PRB4 P10451 OsteopontinSPP1 P10599 Thioredoxin TXN P10600 Transforming growth factor beta-3TGFB3 P10643 Complement component C7 C7 P10645 Vasostatin-2 CHGA P10646Tissue factor pathway inhibitor TFPI P10720 Platelet factor 4variant(4-74) PF4V1 P10745 Retinol-binding protein 3 RBP3 P10767Fibroblast growth factor 6 FGF6 P10909 Clusterin alpha chain CLU P10912Growth hormone receptor GHR P10915 Hyaluronan and proteoglycan linkprotein 1 HAPLN1 P10966 T-cell surface glycoprotein CD8 beta chain CD8BP10997 Islet amyloid polypeptide IAPP P11047 Laminin subunit gamma-1LAMC1 P11150 Hepatic triacylglycerol lipase LIPC P11226 Mannose-bindingprotein C MBL2 P11464 Pregnancy-specific beta-1-glycoprotein 1 PSG1P11465 Pregnancy-specific beta-1-glycoprotein 2 PSG2 P11487 Fibroblastgrowth factor 3 FGF3 P11597 Cholesteryl ester transfer protein CETPP11684 Uteroglobin SCGB1A1 P11686 Pulmonary surfactant-associatedprotein C SFTPC P12034 Fibroblast growth factor 5 FGF5 P12107 Collagenalpha-1(XI) chain COL11A1 P12109 Collagen alpha-1(VI) chain COL6A1P12110 Collagen alpha-2(VI) chain COL6A2 P12111 Collagen alpha-3(VI)chain COL6A3 P12259 Coagulation factor V F5 P12272 PTHrP[1-36] PTHLHP12273 Prolactin-inducible protein PIP P12544 Granzyme A GZMA P12643Bone morphogenetic protein 2 BMP2 P12644 Bone morphogenetic protein 4BMP4 P12645 Bone morphogenetic protein 3 BMP3 P12724 Eosinophil cationicprotein RNASE3 P12821 Angiotensin-converting enzyme, soluble form ACEP12838 Neutrophil defensin 4 DEFA4 P12872 Motilin MLN P13232Interleukin-7 IL7 P13236 C—C motif chemokine 4 CCL4 P13284Gamma-interferon-inducible lysosomal thiol reductase IFI30 P13500 C—Cmotif chemokine 2 CCL2 P13501 C—C motif chemokine 5 CCL5 P13521Secretogranin-2 SCG2 P13591 Neural cell adhesion molecule 1 NCAM1 P13611Versican core protein VCAN P13671 Complement component C6 C6 P13688Carcinoembryonic antigen-related cell adhesion molecule 1 CEACAM1 P13725Oncostatin-M OSM P13726 Tissue factor F3 P13727 Eosinophil granule majorbasic protein PRG2 P13942 Collagen alpha-2(XI) chain COL11A2 P13987 CD59glycoprotein CD59 P14138 Endothelin-3 EDN3 P14174 Macrophage migrationinhibitory factor MIF P14207 Folate receptor beta FOLR2 P14222Perforin-1 PRF1 P14543 Nidogen-1 NID1 P14555 Phospholipase A2, membraneassociated PLA2G2A P14625 Endoplasmin HSP90B1 P14735 Insulin-degradingenzyme IDE P14778 Interleukin-1 receptor type 1, soluble form IL1R1P14780 82 kDa matrix metalloproteinase-9 MMP9 P15018 Leukemia inhibitoryfactor LIF P15085 Carboxypeptidase A1 CPA1 P15086 Carboxypeptidase BCPB1 P15151 Poliovirus receptor PVR P15169 Carboxypeptidase N catalyticchain CPN1 P15248 Interleukin-9 IL9 P15291 N-acetyllactosamine synthaseB4GALT1 P15309 PAPf39 ACPP P15328 Folate receptor alpha FOLR1 P15374Ubiquitin carboxyl-terminal hydrolase isozyme L3 UCHL3 P15502 ElastinELN P15509 Granulocyte-macrophage colony-stimulating factor receptorsubunit CSF2RA alpha P15515 Histatin-1 HTN1 P15516 His3-(31-51)-peptideHTN3 P15692 Vascular endothelial growth factor A VEGFA P15814Immunoglobulin lambda-like polypeptide 1 IGLL1 P15907 Beta-galactosidealpha-2,6-sialyltransferase 1 ST6GAL1 P15941 Mucin-1 subunit beta MUC1P16035 Metalloproteinase inhibitor 2 TIMP2 P16112 Aggrecan core protein2 ACAN P16233 Pancreatic triacylglycerol lipase PNLIP P16442 Histo-bloodgroup ABO system transferase ABO P16471 Prolactin receptor PRLR P16562Cysteine-rich secretory protein 2 CRISP2 P16619 C—C motif chemokine3-like 1 CCL3L1 P16860 BNP(3-29) NPPB P16870 Carboxypeptidase E CPEP16871 Interleukin-7 receptor subunit alpha IL7R P17213 Bactericidalpermeability-increasing protein BPI P17538 Chymotrypsinogen B CTRB1P17931 Galectin-3 LGALS3 P17936 Insulin-like growth factor-bindingprotein 3 IGFBP3 P17948 Vascular endothelial growth factor receptor 1FLT1 P18065 Insulin-like growth factor-binding protein 2 IGFBP2 P18075Bone morphogenetic protein 7 BMP7 P18428 Lipopolysaccharide-bindingprotein LBP P18509 PACAP-related peptide ADCYAP1 P18510 Interleukin-1receptor antagonist protein IL1RN P18827 Syndecan-1 SDC1 P19021Peptidylglycine alpha-hydroxylating monooxygenase PAM P19235Erythropoietin receptor EPOR P19438 Tumor necrosis factor-bindingprotein 1 TNFRSF1A P19652 Alpha-1-acid glycoprotein 2 ORM2 P19801Amiloride-sensitive amine oxidase [copper-containing] ABP1 P19823Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 P19827Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 P19835 Bilesalt-activated lipase CEL P19875 C—X—C motif chemokine 2 CXCL2 P19876C—X—C motif chemokine 3 CXCL3 P19883 Follistatin FST P19957 Elafin PI3P19961 Alpha-amylase 2B AMY2B P20061 Transcobalamin-1 TCN1 P20062Transcobalamin-2 TCN2 P20142 Gastricsin PGC P20155 Serine proteaseinhibitor Kazal-type 2 SPINK2 P20231 Tryptase beta-2 TPSB2 P20333 Tumornecrosis factor receptor superfamily member 1B TNFRSF1B P20366 SubstanceP TAC1 P20382 Melanin-concentrating hormone PMCH P20396 Thyroliberin TRHP20742 Pregnancy zone protein PZP P20774 Mimecan OGN P20783Neurotrophin-3 NTF3 P20800 Endothelin-2 EDN2 P20809 Interleukin-11 IL11P20827 Ephrin-A1 EFNA1 P20849 Collagen alpha-1(IX) chain COL9A1 P20851C4b-binding protein beta chain C4BPB P20908 Collagen alpha-1(V) chainCOL5A1 P21128 Poly(U)-specific endoribonuclease ENDOU P21246Pleiotrophin PTN P21583 Kit ligand KITLG P21741 Midkine MDK P21754 Zonapellucida sperm-binding protein 3 ZP3 P21781 Fibroblast growth factor 7FGF7 P21802 Fibroblast growth factor receptor 2 FGFR2 P21810 BiglycanBGN P21815 Bone sialoprotein 2 IBSP P21860 Receptor tyrosine-proteinkinase erbB-3 ERBB3 P21941 Cartilage matrix protein MATN1 P22003 Bonemorphogenetic protein 5 BMP5 P22004 Bone morphogenetic protein 6 BMP6P22079 Lactoperoxidase LPO P22105 Tenascin-X TNXB P22301 Interleukin-10IL10 P22303 Acetylcholinesterase ACHE P22352 Glutathione peroxidase 3GPX3 P22362 C—C motif chemokine 1 CCL1 P22455 Fibroblast growth factorreceptor 4 FGFR4 P22466 Galanin message-associated peptide GAL P22692Insulin-like growth factor-binding protein 4 IGFBP4 P22749 GranulysinGNLY P22792 Carboxypeptidase N subunit 2 CPN2 P22891 Vitamin K-dependentprotein Z PROZ P22894 Neutrophil collagenase MMP8 P23142 Fibulin-1 FBLN1P23280 Carbonic anhydrase 6 CA6 P23352 Anosmin-1 KAL1 P23435Cerebellin-1 CBLN1 P23560 Brain-derived neurotrophic factor BDNF P23582C-type natriuretic peptide NPPC P23946 Chymase CMA1 P24043 Lamininsubunit alpha-2 LAMA2 P24071 Immunoglobulin alpha Fc receptor FCARP24347 Stromelysin-3 MMP11 P24387 Corticotropin-releasing factor-bindingprotein CRHBP P24592 Insulin-like growth factor-binding protein 6 IGFBP6P24593 Insulin-like growth factor-binding protein 5 IGFBP5 P24821Tenascin TNC P24855 Deoxyribonuclease-1 DNASE1 P25067 Collagenalpha-2(VIII) chain COL8A2 P25311 Zinc-alpha-2-glycoprotein AZGP1 P25391Laminin subunit alpha-1 LAMA1 P25445 Tumor necrosis factor receptorsuperfamily member 6 FAS P25940 Collagen alpha-3(V) chain COL5A3 P25942Tumor necrosis factor receptor superfamily member 5 CD40 P26022Pentraxin-related protein PTX3 PTX3 P26927 Hepatocyte growth factor-likeprotein beta chain MST1 P27169 Serum paraoxonase/arylesterase 1 PON1P27352 Gastric intrinsic factor GIF P27487 Dipeptidyl peptidase 4membrane form DPP4 P27539 Embryonic growth/differentiation factor 1 GDF1P27658 Vastatin COL8A1 P27797 Calreticulin CALR P27918 Properdin CFPP28039 Acyloxyacyl hydrolase AOAH P28300 Protein-lysine 6-oxidase LOXP28325 Cystatin-D CST5 P28799 Granulin-1 GRN P29122 Proproteinconvertase subtilisin/kexin type 6 PCSK6 P29279 Connective tissue growthfactor CTGF P29320 Ephrin type-A receptor 3 EPHA3 P29400 Collagenalpha-5(IV) chain COL4A5 P29459 Interleukin-12 subunit alpha IL12AP29460 Interleukin-12 subunit beta IL12B P29508 Serpin B3 SERPINB3P29622 Kallistatin SERPINA4 P29965 CD40 ligand, soluble form CD40LGP30990 Neurotensin/neuromedin N NTS P31025 Lipocalin-1 LCN1 P31151Protein S100-A7 S100A7 P31371 Fibroblast growth factor 9 FGF9 P31431Syndecan-4 SDC4 P31947 14-3-3 protein sigma SFN P32455Interferon-induced guanylate-binding protein 1 GBP1 P32881 Interferonalpha-8 IFNA8 P34096 Ribonuclease 4 RNASE4 P34130 Neurotrophin-4 NTF4P34820 Bone morphogenetic protein 8B BMP8B P35030 Trypsin-3 PRSS3 P35052Secreted glypican-1 GPC1 P35070 Betacellulin BTC P35225 Interleukin-13IL13 P35247 Pulmonary surfactant-associated protein D SFTPD P35318 ADMADM P35542 Serum amyloid A-4 protein SAA4 P35555 Fibrillin-1 FBN1 P35556Fibrillin-2 FBN2 P35625 Metalloproteinase inhibitor 3 TIMP3 P35858Insulin-like growth factor-binding protein complex acid labile subunitIGFALS P35916 Vascular endothelial growth factor receptor 3 FLT4 P35968Vascular endothelial growth factor receptor 2 KDR P36222Chitinase-3-like protein 1 CHI3L1 P36952 Serpin B5 SERPINB5 P36955Pigment epithelium-derived factor SERPINF1 P36980 Complement factorH-related protein 2 CFHR2 P39059 Collagen alpha-1(XV) chain COL15A1P39060 Collagen alpha-1(XVIII) chain COL18A1 P39877 Calcium-dependentphospholipase A2 PLA2G5 P39900 Macrophage metalloelastase MMP12 P39905Glial cell line-derived neurotrophic factor GDNF P40225 ThrombopoietinTHPO P40967 M-alpha PMEL P41159 Leptin LEP P41221 Protein Wnt-5a WNT5AP41222 Prostaglandin-H2 D-isomerase PTGDS P41271 Neuroblastomasuppressor of tumorigenicity 1 NBL1 P41439 Folate receptor gamma FOLR3P42127 Agouti-signaling protein ASIP P42702 Leukemia inhibitory factorreceptor LIFR P42830 ENA-78(9-78) CXCL5 P43026 Growth/differentiationfactor 5 GDF5 P43251 Biotinidase BTD P43652 Afamin AFM P45452Collagenase 3 MMP13 P47710 Casoxin-D CSN1S1 P47929 Galectin-7 LGALS7BP47972 Neuronal pentraxin-2 NPTX2 P47989 Xanthine oxidase XDH P47992Lymphotactin XCL1 P48023 Tumor necrosis factor ligand superfamily member6, membrane FASLG form P48052 Carboxypeptidase A2 CPA2 P48061 Stromalcell-derived factor 1 CXCL12 P48304 Lithostathine-1-beta REG1B P48307Tissue factor pathway inhibitor 2 TFPI2 P48357 Leptin receptor LEPRP48594 Serpin B4 SERPINB4 P48645 Neuromedin-U-25 NMU P48740Mannan-binding lectin serine protease 1 MASP1 P48745 Protein NOV homologNOV P48960 CD97 antigen subunit beta CD97 P49223 Kunitz-type proteaseinhibitor 3 SPINT3 P49747 Cartilage oligomeric matrix protein COMPP49763 Placenta growth factor PGF P49765 Vascular endothelial growthfactor B VEGFB P49767 Vascular endothelial growth factor C VEGFC P49771Fms-related tyrosine kinase 3 ligand FLT3LG P49862 Kallikrein-7 KLK7P49863 Granzyme K GZMK P49908 Selenoprotein P SEPP1 P49913 Antibacterialprotein FALL-39 CAMP P50607 Tubby protein homolog TUB P51124 Granzyme MGZMM P51512 Matrix metalloproteinase-16 MMP16 P51654 Glypican-3 GPC3P51671 Eotaxin CCL11 P51884 Lumican LUM P51888 Prolargin PRELP P52798Ephrin-A4 EFNA4 P52823 Stanniocalcin-1 STC1 P53420 Collagen alpha-4(IV)chain COL4A4 P53621 Coatomer subunit alpha COPA P54108 Cysteine-richsecretory protein 3 CRISP3 P54315 Pancreatic lipase-related protein 1PNLIPRP1 P54317 Pancreatic lipase-related protein 2 PNLIPRP2 P54793Arylsulfatase F ARSF P55000 Secreted Ly-6/uPAR-related protein 1 SLURP1P55001 Microfibrillar-associated protein 2 MFAP2 P55056 ApolipoproteinC-IV APOC4 P55058 Phospholipid transfer protein PLTP P55075 Fibroblastgrowth factor 8 FGF8 P55081 Microfibrillar-associated protein 1 MFAP1P55083 Microfibril-associated glycoprotein 4 MFAP4 P55107 Bonemorphogenetic protein 3B GDF10 P55145 Mesencephalic astrocyte-derivedneurotrophic factor MANF P55259 Pancreatic secretory granule membranemajor glycoprotein GP2 GP2 P55268 Laminin subunit beta-2 LAMB2 P55773CCL23(30-99) CCL23 P55774 C—C motif chemokine 18 CCL18 P55789 FAD-linkedsulfhydryl oxidase ALR GFER P56703 Proto-oncogene Wnt-3 WNT3 P56704Protein Wnt-3a WNT3A P56705 Protein Wnt-4 WNT4 P56706 Protein Wnt-7bWNT7B P56730 Neurotrypsin PRSS12 P56851 Epididymal secretory proteinE3-beta EDDM3B P56975 Neuregulin-3 NRG3 P58062 Serine protease inhibitorKazal-type 7 SPINK7 P58215 Lysyl oxidase homolog 3 LOXL3 P58294Prokineticin-1 PROK1 P58335 Anthrax toxin receptor 2 ANTXR2 P58397 Adisintegrin and metalloproteinase with thrombospondin motifs 12 ADAMTS12P58417 Neurexophilin-1 NXPH1 P58499 Protein FAM3B FAM3B P59510 Adisintegrin and metalloproteinase with thrombospondin motifs 20 ADAMTS20P59665 Neutrophil defensin 1 DEFA1B P59666 Neutrophil defensin 3 DEFA3P59796 Glutathione peroxidase 6 GPX6 P59826 BPI fold-containing family Bmember 3 BPIFB3 P59827 BPI fold-containing family B member 4 BPIFB4P59861 Beta-defensin 131 DEFB131 P60022 Beta-defensin 1 DEFB1 P60153Inactive ribonuclease-like protein 9 RNASE9 P60827 Complement C1q tumornecrosis factor-related protein 8 C1QTNF8 P60852 Zona pellucidasperm-binding protein 1 ZP1 P60985 Keratinocytedifferentiation-associated protein KRTDAP P61109 Kidneyandrogen-regulated protein KAP P61278 Somatostatin-14 SST P61366Osteocrin OSTN P61626 Lysozyme C LYZ P61769 Beta-2-microglobulin B2MP61812 Transforming growth factor beta-2 TGFB2 P61916 Epididymalsecretory protein E1 NPC2 P62502 Epididymal-specific lipocalin-6 LCN6P62937 Peptidyl-prolyl cis-trans isomerase A PPIA P67809Nuclease-sensitive element-binding protein 1 YBX1 P67812 Signalpeptidase complex catalytic subunit SEC11A SEC11A P78310 Coxsackievirusand adenovirus receptor CXADR P78333 Secreted glypican-5 GPC5 P78380Oxidized low-density lipoprotein receptor 1 OLR1 P78423 Processedfractalkine CX3CL1 P78509 Reelin RELN P78556 CCL20(2-70) CCL20 P80075MCP-2(6-76) CCL8 P80098 C—C motif chemokine 7 CCL7 P80108Phosphatidylinositol-glycan-specific phospholipase D GPLD1 P80162 C—X—Cmotif chemokine 6 CXCL6 P80188 Neutrophil gelatinase-associatedlipocalin LCN2 P80303 Nucleobindin-2 NUCB2 P80511 Calcitermin S100A12P81172 Hepcidin-25 HAMP P81277 Prolactin-releasing peptide PRLH P81534Beta-defensin 103 DEFB103A P81605 Dermcidin DCD P82279 Protein crumbshomolog 1 CRB1 P82987 ADAMTS-like protein 3 ADAMTSL3 P83105 Serineprotease HTRA4 HTRA4 P83110 Serine protease HTRA3 HTRA3 P83859Orexigenic neuropeptide QRFP QRFP P98088 Mucin-5AC MUC5AC P98095Fibulin-2 FBLN2 P98160 Basement membrane-specific heparan sulfateproteoglycan core HSPG2 protein P98173 Protein FAM3A FAM3A Q00604 NorrinNDP Q00796 Sorbitol dehydrogenase SORD Q00887 Pregnancy-specificbeta-1-glycoprotein 9 PSG9 Q00888 Pregnancy-specific beta-1-glycoprotein4 PSG4 Q00889 Pregnancy-specific beta-1-glycoprotein 6 PSG6 Q01523HD5(56-94) DEFA5 Q01524 Defensin-6 DEFA6 Q01955 Collagen alpha-3(IV)chain COL4A3 Q02297 Pro-neuregulin-1, membrane-bound isoform NRG1 Q02325Plasminogen-like protein B PLGLB1 Q02383 Semenogelin-2 SEMG2 Q02388Collagen alpha-1(VII) chain COL7A1 Q02505 Mucin-3A MUC3A Q02509Otoconin-90 OC90 Q02747 Guanylin GUCA2A Q02763 Angiopoietin-1 receptorTEK Q02817 Mucin-2 MUC2 Q02985 Complement factor H-related protein 3CFHR3 Q03167 Transforming growth factor beta receptor type 3 TGFBR3Q03403 Trefoil factor 2 TFF2 Q03405 Urokinase plasminogen activatorsurface receptor PLAUR Q03591 Complement factor H-related protein 1CFHR1 Q03692 Collagen alpha-1(X) chain COL10A1 Q04118 Basic salivaryproline-rich protein 3 PRB3 Q04756 Hepatocyte growth factor activatorshort chain HGFAC Q04900 Sialomucin core protein 24 CD164 Q05315Eosinophil lysophospholipase CLC Q05707 Collagen alpha-1(XIV) chainCOL14A1 Q05996 Processed zona pellucida sperm-binding protein 2 ZP2Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 Q06141Regenerating islet-derived protein 3-alpha REG3A Q06828 FibromodulinFMOD Q07092 Collagen alpha-1(XVI) chain COL16A1 Q07325 C—X—C motifchemokine 9 CXCL9 Q07507 Dermatopontin DPT Q075Z2 Binder of spermprotein homolog 1 BSPH1 Q07654 Trefoil factor 3 TFF3 Q07699 Sodiumchannel subunit beta-1 SCN1B Q08345 Epithelial discoidindomain-containing receptor 1 DDR1 Q08380 Galectin-3-binding proteinLGALS3BP Q08397 Lysyl oxidase homolog 1 LOXL1 Q08431 Lactadherin MFGE8Q08629 Testican-1 SPOCK1 Q08648 Sperm-associated antigen 11B SPAG11BQ08830 Fibrinogen-like protein 1 FGL1 Q10471 PolypeptideN-acetylgalactosaminyltransferase 2 GALNT2 Q10472 PolypeptideN-acetylgalactosaminyltransferase 1 GALNT1 Q11201CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3- ST3GAL1sialyltransferase 1 Q11203 CMP-N-acetylneuraminate-beta-1,4-galactosidealpha-2,3- ST3GAL3 sialyltransferase Q11206CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3- ST3GAL4sialyltransferase 4 Q12794 Hyaluronidase-1 HYAL1 Q12805 EGF-containingfibulin-like extracellular matrix protein 1 EFEMP1 Q12836 Zona pellucidasperm-binding protein 4 ZP4 Q12841 Follistatin-related protein 1 FSTL1Q12904 Aminoacyl tRNA synthase complex-interacting multifunctional AIMP1protein 1 Q13018 Soluble secretory phospholipase A2 receptor PLA2R1Q13072 B melanoma antigen 1 BAGE Q13093 Platelet-activating factoracetylhydrolase PLA2G7 Q13103 Secreted phosphoprotein 24 SPP2 Q13162Peroxiredoxin-4 PRDX4 Q13201 Platelet glycoprotein Ia* MMRN1 Q13214Semaphorin-3B SEMA3B Q13219 Pappalysin-1 PAPPA Q13231 Chitotriosidase-1CHIT1 Q13253 Noggin NOG Q13261 Interleukin-15 receptor subunit alphaIL15RA Q13275 Semaphorin-3F SEMA3F Q13291 Signaling lymphocyticactivation molecule SLAMF1 Q13316 Dentin matrix acidic phosphoprotein 1DMP1 Q13361 Microfibrillar-associated protein 5 MFAP5 Q13410Butyrophilin subfamily 1 member A1 BTN1A1 Q13421 Mesothelin, cleavedform MSLN Q13429 Insulin-like growth factor I IGF-I Q13443 Disintegrinand metalloproteinase domain-containing protein 9 ADAM9 Q13519Neuropeptide 1 PNOC Q13751 Laminin subunit beta-3 LAMB3 Q13753 Lamininsubunit gamma-2 LAMC2 Q13790 Apolipoprotein F APOF Q13822 Ectonucleotidepyrophosphatase/phosphodiesterase family member 2 ENPP2 Q14031 Collagenalpha-6(IV) chain COL4A6 Q14050 Collagen alpha-3(IX) chain COL9A3 Q14055Collagen alpha-2(IX) chain COL9A2 Q14112 Nidogen-2 NID2 Q14114Low-density lipoprotein receptor-related protein 8 LRP8 Q14118Dystroglycan DAG1 Q14314 Fibroleukin FGL2 Q14393 Growth arrest-specificprotein 6 GAS6 Q14406 Chorionic somatomammotropin hormone-like 1 CSHL1Q14507 Epididymal secretory protein E3-alpha EDDM3A Q14508 WAPfour-disulfide core domain protein 2 WFDC2 Q14512 Fibroblast growthfactor-binding protein 1 FGFBP1 Q14515 SPARC-like protein 1 SPARCL1Q14520 Hyaluronan-binding protein 2 27 kDa light chain HABP2 Q14563Semaphorin-3A SEMA3A Q14623 Indian hedgehog protein IHH Q14624Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 Q14667 UPF0378protein KIAA0100 KIAA0100 Q14703 Membrane-bound transcription factorsite-1 protease MBTPS1 Q14766 Latent-transforming growth factorbeta-binding protein 1 LTBP1 Q14767 Latent-transforming growth factorbeta-binding protein 2 LTBP2 Q14773 Intercellular adhesion molecule 4ICAM4 Q14993 Collagen alpha-1(XIX) chain COL19A1 Q14CN2Calcium-activated chloride channel regulator 4, 110 kDa form CLCA4Q15046 Lysine--tRNA ligase KARS Q15063 Periostin POSTN Q15109 Advancedglycosylation end product-specific receptor AGER Q15113 ProcollagenC-endopeptidase enhancer 1 PCOLCE Q15166 Serum paraoxonase/lactonase 3PON3 Q15195 Plasminogen-like protein A PLGLA Q15198 Platelet-derivedgrowth factor receptor-like protein PDGFRL Q15223 Poliovirusreceptor-related protein 1 PVRL1 Q15238 Pregnancy-specificbeta-1-glycoprotein 5 PSG5 Q15363 Transmembrane emp24 domain-containingprotein 2 TMED2 Q15375 Ephrin type-A receptor 7 EPHA7 Q15389Angiopoietin-1 ANGPT1 Q15465 Sonic hedgehog protein SHH Q15485 Ficolin-2FCN2 Q15517 Corneodesmosin CDSN Q15582 Transforming growthfactor-beta-induced protein ig-h3 TGFBI Q15661 Tryptase alpha/beta-1TPSAB1 Q15726 Metastin KISS1 Q15782 Chitinase-3-like protein 2 CHI3L2Q15828 Cystatin-M CST6 Q15846 Clusterin-like protein 1 CLUL1 Q15848Adiponectin ADIPOQ Q16206 Protein disulfide-thiol oxidoreductase ENOX2Q16270 Insulin-like growth factor-binding protein 7 IGFBP7 Q16363Laminin subunit alpha-4 LAMA4 Q16378 Proline-rich protein 4 PRR4 Q16557Pregnancy-specific beta-1-glycoprotein 3 PSG3 Q16568 CART(42-89) CARTPTQ16610 Extracellular matrix protein 1 ECM1 Q16619 Cardiotrophin-1 CTF1Q16623 Syntaxin-1A STX1A Q16627 HCC-1(9-74) CCL14 Q16651 Prostasin lightchain PRSS8 Q16661 Guanylate cyclase C-activating peptide 2 GUCA2BQ16663 CCL15(29-92) CCL15 Q16674 Melanoma-derived growth regulatoryprotein MIA Q16769 Glutaminyl-peptide cyclotransferase QPCT Q16787Laminin subunit alpha-3 LAMA3 Q16842CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3- ST3GAL2sialyltransferase 2 Q17RR3 Pancreatic lipase-related protein 3 PNLIPRP3Q17RW2 Collagen alpha-1(XXIV) chain COL24A1 Q17RY6 Lymphocyte antigen 6KLY6K Q1L6U9 Prostate-associated microseminoprotein MSMP Q1W4C9 Serineprotease inhibitor Kazal-type 13 SPINK13 Q1ZYL8 Izumo sperm-egg fusionprotein 4 IZUMO4 Q29960 HLA class I histocompatibility antigen, Cw-16alpha chain HLA-C Q2I0M5 R-spondin-4 RSPO4 Q2L4Q9 Serine protease 53PRSS53 Q2MKA7 R-spondin-1 RSPO1 Q2MV58 Tectonic-1 TCTN1 Q2TAL6 BrorinVWC2 Q2UY09 Collagen alpha-1(XXVIII) chain COL28A1 Q2VPA4 Complementcomponent receptor 1-like protein CR1L Q2WEN9 Carcinoembryonicantigen-related cell adhesion molecule 16 CEACAM16 Q30KP8 Beta-defensin136 DEFB136 Q30KP9 Beta-defensin 135 DEFB135 Q30KQ1 Beta-defensin 133DEFB133 Q30KQ2 Beta-defensin 130 DEFB130 Q30KQ4 Beta-defensin 116DEFB116 Q30KQ5 Beta-defensin 115 DEFB115 Q30KQ6 Beta-defensin 114DEFB114 Q30KQ7 Beta-defensin 113 DEFB113 Q30KQ8 Beta-defensin 112DEFB112 Q30KQ9 Beta-defensin 110 DEFB110 Q30KR1 Beta-defensin 109DEFB109P1 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 Q3B7J2 Glucose-fructoseoxidoreductase domain-containing protein 2 GFOD2 Q3SY79 Protein WntWNT3A Q3T906 N-acetylglucosamine-1-phosphotransferase subunitsalpha/beta GNPTAB Q495T6 Membrane metallo-endopeptidase-like 1 MMEL1Q49AH0 Cerebral dopamine neurotrophic factor CDNF Q4G0G5 Secretoglobinfamily 2B member 2 SCGB2B2 Q4G0M1 Protein FAM132B FAM132B Q4LDE5 Sushi,von Willebrand factor type A, EGF and pentraxin domain- SVEP1 containingprotein 1 Q4QY38 Beta-defensin 134 DEFB134 Q4VAJ4 Protein Wnt WNT10BQ4W5P6 Protein TMEM155 TMEM155 Q4ZHG4 Fibronectin type IIIdomain-containing protein 1 FNDC1 Q53H76 Phospholipase A1 member A PLA1AQ53RD9 Fibulin-7 FBLN7 Q53S33 BolA-like protein 3 BOLA3 Q5BLP8Neuropeptide-like protein C4orf48 C4orf48 Q5DT21 Serine proteaseinhibitor Kazal-type 9 SPINK9 Q5EBL8 PDZ domain-containing protein 11PDZD11 Q5FYB0 Arylsulfatase J ARSJ Q5FYB1 Arylsulfatase I ARSI Q5GAN3Ribonuclease-like protein 13 RNASE13 Q5GAN4 Ribonuclease-like protein 12RNASE12 Q5GAN6 Ribonuclease-like protein 10 RNASE10 Q5GFL6 vonWillebrand factor A domain-containing protein 2 VWA2 Q5H8A3 Neuromedin-SNMS Q5H8C1 FRAS1-related extracellular matrix protein 1 FREM1 Q5IJ48Protein crumbs homolog 2 CRB2 Q5J5C9 Beta-defensin 121 DEFB121 Q5JS37NHL repeat-containing protein 3 NHLRC3 Q5JTB6 Placenta-specific protein9 PLAC9 Q5JU69 Torsin-2A TOR2A Q5JXM2 Methyltransferase-like protein 24METTL24 Q5JZY3 Ephrin type-A receptor 10 EPHA10 Q5K4E3 Polyserase-2PRSS36 Q5SRR4 Lymphocyte antigen 6 complex locus protein G5c LY6G5CQ5T1H1 Protein eyes shut homolog EYS Q5T4F7 Secreted frizzled-relatedprotein 5 SFRP5 Q5T4W7 Artemin ARTN Q5T7M4 Protein FAM132A FAM132AQ5TEH8 Protein Wnt WNT2B Q5TIE3 von Willebrand factor Adomain-containing protein 5B1 VWA5B1 Q5UCC4 ER membrane protein complexsubunit 10 EMC10 Q5VST6 Abhydrolase domain-containing protein FAM108B1FAM108B1 Q5VTL7 Fibronectin type III domain-containing protein 7 FNDC7Q5VUM1 UPF0369 protein C6orf57 C6orf57 Q5VV43 Dyslexia-associatedprotein KIAA0319 KIAA0319 Q5VWW1 Complement C1q-like protein 3 C1QL3Q5VXI9 Lipase member N LIPN Q5VXJ0 Lipase member K LIPK Q5VXM1 CUBdomain-containing protein 2 CDCP2 Q5VYX0 Renalase RNLS Q5VYY2 Lipasemember M LIPM Q5W186 Cystatin-9 CST9 Q5W5W9 Regulated endocrine-specificprotein 18 RESP18 Q5XG92 Carboxylesterase 4A CES4A Q63HQ2 PikachurinEGFLAM Q641Q3 Meteorin-like protein METRNL Q66K79 Carboxypeptidase Z CPZQ685J3 Mucin-17 MUC17 Q68BL7 Olfactomedin-like protein 2A OLFML2A Q68BL8Olfactomedin-like protein 2B OLFML2B Q68DV7 E3 ubiquitin-protein ligaseRNF43 RNF43 Q6B9Z1 Insulin growth factor-like family member 4 IGFL4Q6BAA4 Fc receptor-like B FCRLB Q6E0U4 Dermokine DMKN Q6EMK4 VasorinVASN Q6FHJ7 Secreted frizzled-related protein 4 SFRP4 Q6GPI1Chymotrypsin B2 chain B CTRB2 Q6GTS8 Probable carboxypeptidase PM20D1PM20D1 Q6H9L7 Isthmin-2 ISM2 Q6IE36 Ovostatin homolog 2 OVOS2 Q6IE37Ovostatin homolog 1 OVOS1 Q6IE38 Serine protease inhibitor Kazal-type 14SPINK14 Q6ISS4 Leukocyte-associated immunoglobulin-like receptor 2 LAIR2Q6JVE5 Epididymal-specific lipocalin-12 LCN12 Q6JVE6 Epididymal-specificlipocalin-10 LCN10 Q6JVE9 Epididymal-specific lipocalin-8 LCN8 Q6KF10Growth/differentiation factor 6 GDF6 Q6MZW2 Follistatin-related protein4 FSTL4 Q6NSX1 Coiled-coil domain-containing protein 70 CCDC70 Q6NT32Carboxylesterase 5A CES5A Q6NT52 Choriogonadotropin subunit beta variant2 CGB2 Q6NUI6 Chondroadherin-like protein CHADL Q6NUJ1 Saposin A-likePSAPL1 Q6P093 Arylacetamide deacetylase-like 2 AADACL2 Q6P4A8Phospholipase B-like 1 PLBD1 Q6P5S2 UPF0762 protein C6orf58 C6orf58Q6P988 Protein notum homolog NOTUM Q6PCB0 von Willebrand factor Adomain-containing protein 1 VWA1 Q6PDA7 Sperm-associated antigen 11ASPAG11A Q6PEW0 Inactive serine protease 54 PRSS54 Q6PEZ8 Podocan-likeprotein 1 PODNL1 Q6PKH6 Dehydrogenase/reductase SDR family member 4-like2 DHRS4L2 Q6Q788 Apolipoprotein A-V APOA5 Q6SPF0 Atherin SAMD1 Q6UDR6Kunitz-type protease inhibitor 4 SPINT4 Q6URK8 Testis, prostate andplacenta-expressed protein TEPP Q6UW01 Cerebellin-3 CBLN3 Q6UW10Surfactant-associated protein 2 SFTA2 Q6UW15 Regenerating islet-derivedprotein 3-gamma REG3G Q6UW32 Insulin growth factor-like family member 1IGFL1 Q6UW78 UPF0723 protein C11orf83 C11orf83 Q6UW88 Epigen EPGN Q6UWE3Colipase-like protein 2 CLPSL2 Q6UWF7 NXPE family member 4 NXPE4 Q6UWF9Protein FAM180A FAM180A Q6UWM5 GLIPR1-like protein 1 GLIPR1L1 Q6UWN8Serine protease inhibitor Kazal-type 6 SPINK6 Q6UWP2Dehydrogenase/reductase SDR family member 11 DHRS11 Q6UWP8 SuprabasinSBSN Q6UWQ5 Lysozyme-like protein 1 LYZL1 Q6UWQ7 Insulin growthfactor-like family member 2 IGFL2 Q6UWR7 Ectonucleotidepyrophosphatase/phosphodiesterase family member ENPP6 6 soluble formQ6UWT2 Adropin ENHO Q6UWU2 Beta-galactosidase-1-like protein GLB1LQ6UWW0 Lipocalin-15 LCN15 Q6UWX4 HHIP-like protein 2 HHIPL2 Q6UWY0Arylsulfatase K ARSK Q6UWY2 Serine protease 57 PRSS57 Q6UWY5Olfactomedin-like protein 1 OLFML1 Q6UX06 Olfactomedin-4 OLFM4 Q6UX07Dehydrogenase/reductase SDR family member 13 DHRS13 Q6UX39 Amelotin AMTNQ6UX46 Protein FAM150B FAM150B Q6UX73 UPF0764 protein C16orf89 C16orf89Q6UXB0 Protein FAM131A FAM131A Q6UXB1 Insulin growth factor-like familymember 3 IGFL3 Q6UXB2 VEGF co-regulated chemokine 1 CXCL17 Q6UXF7 C-typelectin domain family 18 member B CLEC18B Q6UXH0 Hepatocellularcarcinoma-associated protein TD26 C19orf80 Q6UXH1 Cysteine-rich withEGF-like domain protein 2 CRELD2 Q6UXH8 Collagen and calcium-binding EGFdomain-containing protein 1 CCBE1 Q6UXH9 Inactive serine protease PAMR1PAMR1 Q6UXI7 Vitrin VIT Q6UXI9 Nephronectin NPNT Q6UXN2 Trem-liketranscript 4 protein TREML4 Q6UXS0 C-type lectin domain family 19 memberA CLEC19A Q6UXT8 Protein FAM150A FAM150A Q6UXT9 Abhydrolasedomain-containing protein 15 ABHD15 Q6UXV4 Apolipoprotein O-like APOOLQ6UXX5 Inter-alpha-trypsin inhibitor heavy chain H6 ITIH6 Q6UXX9R-spondin-2 RSPO2 Q6UY14 ADAMTS-like protein 4 ADAMTSL4 Q6UY27 Prostateand testis expressed protein 2 PATE2 Q6W4X9 Mucin-6 MUC6 Q6WN34Chordin-like protein 2 CHRDL2 Q6WRI0 Immunoglobulin superfamily member10 IGSF10 Q6X4U4 Sclerostin domain-containing protein 1 SOSTDC1 Q6X784Zona pellucida-binding protein 2 ZPBP2 Q6XE38 Secretoglobin family 1Dmember 4 SCGB1D4 Q6XPR3 Repetin RPTN Q6XZB0 Lipase member I LIPI Q6ZMM2ADAMTS-like protein 5 ADAMTSL5 Q6ZMP0 Thrombospondin type-1domain-containing protein 4 THSD4 Q6ZNF0 Iron/zinc purple acidphosphatase-like protein PAPL Q6ZRI0 Otogelin OTOG Q6ZRP7 Sulfhydryloxidase 2 QSOX2 Q6ZWJ8 Kielin/chordin-like protein KCP Q75N90Fibrillin-3 FBN3 Q765I0 Urotensin-2B UTS2D Q76B58 Protein FAM5C FAM5CQ76LX8 A disintegrin and metalloproteinase with thrombospondin motifs 13ADAMTS13 Q76M96 Coiled-coil domain-containing protein 80 CCDC80 Q7L1S5Carbohydrate sulfotransferase 9 CHST9 Q7L513 Fc receptor-like A FCRLAQ7L8A9 Vasohibin-1 VASH1 Q7RTM1 Otopetrin-1 OTOP1 Q7RTW8 Otoancorin OTOAQ7RTY5 Serine protease 48 PRSS48 Q7RTY7 Ovochymase-1 OVCH1 Q7RTZ1Ovochymase-2 OVCH2 Q7Z304 MAM domain-containing protein 2 MAMDC2 Q7Z3S9Notch homolog 2 N-terminal-like protein NOTCH2NL Q7Z4H4 Intermedin-shortADM2 Q7Z4P5 Growth/differentiation factor 7 GDF7 Q7Z4R8 UPF0669 proteinC6orf120 C6orf120 Q7Z4W2 Lysozyme-like protein 2 LYZL2 Q7Z5A4 Serineprotease 42 PRSS42 Q7Z5A7 Protein FAM19A5 FAM19A5 Q7Z5A8 Protein FAM19A3FAM19A3 Q7Z5A9 Protein FAM19A1 FAM19A1 Q7Z5J1 Hydroxysteroid11-beta-dehydrogenase 1-like protein HSD11B1L Q7Z5L0 Vitelline membraneouter layer protein 1 homolog VMO1 Q7Z5L3 Complement C1q-like protein 2C1QL2 Q7Z5L7 Podocan PODN Q7Z5P4 17-beta-hydroxysteroid dehydrogenase 13HSD17B13 Q7Z5P9 Mucin-19 MUC19 Q7Z5Y6 Bone morphogenetic protein 8ABMP8A Q7Z7B7 Beta-defensin 132 DEFB132 Q7Z7B8 Beta-defensin 128 DEFB128Q7Z7C8 Transcription initiation factor TFIID subunit 8 TAF8 Q7Z7H5Transmembrane emp24 domain-containing protein 4 TMED4 Q86SG7 Lysozymeg-like protein 2 LYG2 Q86SI9 Protein CEI C5orf38 Q86TE4 Leucine zipperprotein 2 LUZP2 Q86TH1 ADAMTS-like protein 2 ADAMTSL2 Q86U17 Serpin A11SERPINA11 Q86UU9 Endokinin-A TAC4 Q86UW8 Hyaluronan and proteoglycanlink protein 4 HAPLN4 Q86UX2 Inter-alpha-trypsin inhibitor heavy chainH5 ITIH5 Q86V24 Adiponectin receptor protein 2 ADIPOR2 Q86VB7 SolubleCD163 CD163 Q86VR8 Four-jointed box protein 1 FJX1 Q86WD7 Serpin A9SERPINA9 Q86WN2 Interferon epsilon IFNE Q86WS3 Placenta-specific 1-likeprotein PLAC1L Q86X52 Chondroitin sulfate synthase 1 CHSY1 Q86XP6Gastrokine-2 GKN2 Q86XS5 Angiopoietin-related protein 5 ANGPTL5 Q86Y27 Bmelanoma antigen 5 BAGE5 Q86Y28 B melanoma antigen 4 BAGE4 Q86Y29 Bmelanoma antigen 3 BAGE3 Q86Y30 B melanoma antigen 2 BAGE2 Q86Y38Xylosyltransferase 1 XYLT1 Q86Y78 Ly6/PLAUR domain-containing protein 6LYPD6 Q86YD3 Transmembrane protein 25 TMEM25 Q86YJ6 Threoninesynthase-like 2 THNSL2 Q86YW7 Glycoprotein hormone beta-5 GPHB5 Q86Z23Complement C1q-like protein 4 C1QL4 Q8IU57 Interleukin-28 receptorsubunit alpha IL28RA Q8IUA0 WAP four-disulfide core domain protein 8WFDC8 Q8IUB2 WAP four-disulfide core domain protein 3 WFDC3 Q8IUB3Protein WFDC10B WFDC10B Q8IUB5 WAP four-disulfide core domain protein 13WFDC13 Q8IUH2 Protein CREG2 CREG2 Q8IUK5 Plexin domain-containingprotein 1 PLXDC1 Q8IUL8 Cartilage intermediate layer protein 2 C2 CILP2Q8IUX7 Adipocyte enhancer-binding protein 1 AEBP1 Q8IUX8 Epidermalgrowth factor-like protein 6 EGFL6 Q8IVL8 Carboxypeptidase O CPO Q8IVN8Somatomedin-B and thrombospondin type-1 domain-containing SBSPON proteinQ8IVW8 Protein spinster homolog 2 SPNS2 Q8IW75 Serpin A12 SERPINA12Q8IW92 Beta-galactosidase-1-like protein 2 GLB1L2 Q8IWL1 Pulmonarysurfactant-associated protein A2 SFTPA2 Q8IWL2 Pulmonarysurfactant-associated protein A1 SFTPA1 Q8IWV2 Contactin-4 CNTN4 Q8IWY4Signal peptide, CUB and EGF-like domain-containing protein 1 SCUBE1Q8IX30 Signal peptide, CUB and EGF-like domain-containing protein 3SCUBE3 Q8IXA5 Sperm acrosome membrane-associated protein 3, membraneform SPACA3 Q8IXB1 DnaJ homolog subfamily C member 10 DNAJC10 Q8IXL6Extracellular serine/threonine protein kinase Fam20C FAM20C Q8IYD9 Lungadenoma susceptibility protein 2 LAS2 Q8IYP2 Serine protease 58 PRSS58Q8IYS5 Osteoclast-associated immunoglobulin-like receptor OSCAR Q8IZC6Collagen alpha-1(XXVII) chain COL27A1 Q8IZJ3 C3 and PZP-likealpha-2-macroglobulin domain-containing protein 8 CPAMD8 Q8IZN7Beta-defensin 107 DEFB107B Q8N0V4 Leucine-rich repeat LGI family member2 LGI2 Q8N104 Beta-defensin 106 DEFB106B Q8N119 Matrixmetalloproteinase-21 MMP21 Q8N129 Protein canopy homolog 4 CNPY4 Q8N135Leucine-rich repeat LGI family member 4 LGI4 Q8N145 Leucine-rich repeatLGI family member 3 LGI3 Q8N158 Glypican-2 GPC2 Q8N1E2 Lysozyme g-likeprotein 1 LYG1 Q8N2E2 von Willebrand factor D and EGF domain-containingprotein VWDE Q8N2E6 Prosalusin TOR2A Q8N2S1 Latent-transforming growthfactor beta-binding protein 4 LTBP4 Q8N302 Angiogenic factor with Gpatch and FHA domains 1 AGGF1 Q8N307 Mucin-20 MUC20 Q8N323 NXPE familymember 1 NXPE1 Q8N387 Mucin-15 MUC15 Q8N3Z0 Inactive serine protease 35PRSS35 Q8N436 Inactive carboxypeptidase-like protein X2 CPXM2 Q8N474Secreted frizzled-related protein 1 SFRP1 Q8N475 Follistatin-relatedprotein 5 FSTL5 Q8N4F0 BPI fold-containing family B member 2 BPIFB2Q8N4T0 Carboxypeptidase A6 CPA6 Q8N5W8 Protein FAM24B FAM24B Q8N687Beta-defensin 125 DEFB125 Q8N688 Beta-defensin 123 DEFB123 Q8N690Beta-defensin 119 DEFB119 Q8N6C5 Immunoglobulin superfamily member 1IGSF1 Q8N6C8 Leukocyte immunoglobulin-like receptor subfamily A member 3LILRA3 Q8N6G6 ADAMTS-like protein 1 ADAMTSL1 Q8N6Y2 Leucine-richrepeat-containing protein 17 LRRC17 Q8N729 Neuropeptide W-23 NPW Q8N8U9BMP-binding endothelial regulator protein BMPER Q8N907 DAN domain familymember 5 DAND5 Q8NAT1 Glycosyltransferase-like domain-containing protein2 GTDC2 Q8NAU1 Fibronectin type III domain-containing protein 5 FNDC5Q8NB37 Parkinson disease 7 domain-containing protein 1 PDDC1 Q8NBI3Draxin DRAXIN Q8NBM8 Prenylcysteine oxidase-like PCYOX1L Q8NBP7Proprotein convertase subtilisin/kexin type 9 PCSK9 Q8NBQ5 Estradiol17-beta-dehydrogenase 11 HSD17B11 Q8NBV8 Synaptotagmin-8 SYT8 Q8NCC3Group XV phospholipase A2 PLA2G15 Q8NCF0 C-type lectin domain family 18member C CLEC18C Q8NCW5 NAD(P)H-hydrate epimerase APOA1BP Q8NDA2Hemicentin-2 HMCN2 Q8NDX9 Lymphocyte antigen 6 complex locus protein G5bLY6G5B Q8NDZ4 Deleted in autism protein 1 C3orf58 Q8NEB7 Acrosin-bindingprotein ACRBP Q8NES8 Beta-defensin 124 DEFB124 Q8NET1 Beta-defensin 108BDEFB108B Q8NEX5 Protein WFDC9 WFDC9 Q8NEX6 Protein WFDC11 WFDC11 Q8NF86Serine protease 33 PRSS33 Q8NFM7 Interleukin-17 receptor D IL17RD Q8NFQ5BPI fold-containing family B member 6 BPIFB6 Q8NFQ6 BPI fold-containingfamily C protein BPIFC Q8NFU4 Follicular dendritic cell secreted peptideFDCSP Q8NFW1 Collagen alpha-1(XXII) chain COL22A1 Q8NG35 Beta-defensin105 DEFB105B Q8NG41 Neuropeptide B-23 NPB Q8NHW6 Otospiralin OTOS Q8NI99Angiopoietin-related protein 6 ANGPTL6 Q8TAA1 Probable ribonuclease 11RNASE11 Q8TAG5 V-set and transmembrane domain-containing protein 2AVSTM2A Q8TAL6 Fin bud initiation factor homolog FIBIN Q8TAT2 Fibroblastgrowth factor-binding protein 3 FGFBP3 Q8TAX7 Mucin-7 MUC7 Q8TB22Spermatogenesis-associated protein 20 SPATA20 Q8TB73 Protein NDNF NDNFQ8TB96 T-cell immunomodulatory protein ITFG1 Q8TC92 Proteindisulfide-thiol oxidoreductase ENOX1 Q8TCV5 WAP four-disulfide coredomain protein 5 WFDC5 Q8TD06 Anterior gradient protein 3 homolog AGR3Q8TD33 Secretoglobin family 1C member 1 SCGB1C1 Q8TD46 Cell surfaceglycoprotein CD200 receptor 1 CD200R1 Q8TDE3 Ribonuclease 8 RNASE8Q8TDF5 Neuropilin and tolloid-like protein 1 NETO1 Q8TDL5 BPIfold-containing family B member 1 BPIFB1 Q8TE56 A disintegrin andmetalloproteinase with thrombospondin motifs 17 ADAMTS17 Q8TE57 Adisintegrin and metalloproteinase with thrombospondin motifs 16 ADAMTS16Q8TE58 A disintegrin and metalloproteinase with thrombospondin motifs 15ADAMTS15 Q8TE59 A disintegrin and metalloproteinase with thrombospondinmotifs 19 ADAMTS19 Q8TE60 A disintegrin and metalloproteinase withthrombospondin motifs 18 ADAMTS18 Q8TE99 Acid phosphatase-like protein 2ACPL2 Q8TER0 Sushi, nidogen and EGF-like domain-containing protein 1SNED1 Q8TEU8 WAP, kazal, immunoglobulin, kunitz and NTRdomain-containing WFIKKN2 protein 2 Q8WTQ1 Beta-defensin 104 DEFB104BQ8WTR8 Netrin-5 NTN5 Q8WTU2 Scavenger receptor cysteine-richdomain-containing group B SRCRB4D protein Q8WU66 Protein TSPEAR TSPEARQ8WUA8 Tsukushin TSKU Q8WUF8 Protein FAM172A FAM172A Q8WUJ1 NeuferricinCYB5D2 Q8WUY1 UPF0670 protein THEM6 THEM6 Q8WVN6 Secreted andtransmembrane protein 1 SECTM1 Q8WVQ1 Soluble calcium-activatednucleotidase 1 CANT1 Q8WWA0 Intelectin-1 ITLN1 Q8WWG1 Neuregulin-4 NRG4Q8WWQ2 Inactive heparanase-2 HPSE2 Q8WWU7 Intelectin-2 ITLN2 Q8WWY7 WAPfour-disulfide core domain protein 12 WFDC12 Q8WWY8 Lipase member H LIPHQ8WWZ8 Oncoprotein-induced transcript 3 protein OIT3 Q8WX39Epididymal-specific lipocalin-9 LCN9 Q8WXA2 Prostate and testisexpressed protein 1 PATE1 Q8WXD2 Secretogranin-3 SCG3 Q8WXF3 Relaxin-3 Achain RLN3 Q8WXI7 Mucin-16 MUC16 Q8WXQ8 Carboxypeptidase A5 CPA5 Q8WXS8A disintegrin and metalloproteinase with thrombospondin motifs 14ADAMTS14 Q92484 Acid sphingomyelinase-like phosphodiesterase 3a SMPDL3AQ92485 Acid sphingomyelinase-like phosphodiesterase 3b SMPDL3B Q92496Complement factor H-related protein 4 CFHR4 Q92520 Protein FAM3C FAM3CQ92563 Testican-2 SPOCK2 Q92583 C—C motif chemokine 17 CCL17 Q92626Peroxidasin homolog PXDN Q92743 Serine protease HTRA1 HTRA1 Q92752Tenascin-R TNR Q92765 Secreted frizzled-related protein 3 FRZB Q92819Hyaluronan synthase 2 HAS2 Q92820 Gamma-glutamyl hydrolase GGH Q92824Proprotein convertase subtilisin/kexin type 5 PCSK5 Q92832 Proteinkinase C-binding protein NELL1 NELL1 Q92838 Ectodysplasin-A, membraneform EDA Q92874 Deoxyribonuclease-1-like 2 DNASE1L2 Q92876 Kallikrein-6KLK6 Q92913 Fibroblast growth factor 13 FGF13 Q92954 Proteoglycan 4C-terminal part PRG4 Q93038 Tumor necrosis factor receptor superfamilymember 25 TNFRSF25 Q93091 Ribonuclease K6 RNASE6 Q93097 Protein Wnt-2bWNT2B Q93098 Protein Wnt-8b WNT8B Q95460 Major histocompatibilitycomplex class I-related gene protein MR1 Q969D9 Thymic stromallymphopoietin TSLP Q969E1 Liver-expressed antimicrobial peptide 2 LEAP2Q969H8 UPF0556 protein C19orf10 C19orf10 Q969Y0 NXPE family member 3NXPE3 Q96A54 Adiponectin receptor protein 1 ADIPOR1 Q96A83 Collagenalpha-1(XXVI) chain EMID2 Q96A84 EMI domain-containing protein 1 EMID1Q96A98 Tuberoinfundibular peptide of 39 residues PTH2 Q96A99 Pentraxin-4PTX4 Q96BH3 Epididymal sperm-binding protein 1 ELSPBP1 Q96BQ1 ProteinFAM3D FAM3D Q96CG8 Collagen triple helix repeat-containing protein 1CTHRC1 Q96DA0 Zymogen granule protein 16 homolog B ZG16B Q96DN2 vonWillebrand factor C and EGF domain-containing protein VWCE Q96DR5 BPIfold-containing family A member 2 BPIFA2 Q96DR8 Mucin-like protein 1MUCL1 Q96DX4 RING finger and SPRY domain-containing protein 1 RSPRY1Q96EE4 Coiled-coil domain-containing protein 126 CCDC126 Q96GS6Abhydrolase domain-containing protein FAM108A1 FAM108A1 Q96GW7 Brevicancore protein BCAN Q96HF1 Secreted frizzled-related protein 2 SFRP2Q96I82 Kazal-type serine protease inhibitor domain-containing protein 1KAZALD1 Q96ID5 Immunoglobulin superfamily member 21 IGSF21 Q96II8Leucine-rich repeat and calponin homology domain-containing LRCH3protein 3 Q96IY4 Carboxypeptidase B2 CPB2 Q96JB6 Lysyl oxidase homolog 4LOXL4 Q96JK4 HHIP-like protein 1 HHIPL1 Q96KN2 Beta-Ala-His dipeptidaseCNDP1 Q96KW9 Protein SPACA7 SPACA7 Q96KX0 Lysozyme-like protein 4 LYZL4Q96L15 Ecto-ADP-ribosyltransferase 5 ART5 Q96LB8 Peptidoglycanrecognition protein 4 PGLYRP4 Q96LB9 Peptidoglycan recognition protein 3PGLYRP3 Q96LC7 Sialic acid-binding Ig-like lectin 10 SIGLEC10 Q96LR4Protein FAM19A4 FAM19A4 Q96MK3 Protein FAM20A FAM20A Q96MS3Glycosyltransferase 1 domain-containing protein 1 GLT1D1 Q96NY8Processed poliovirus receptor-related protein 4 PVRL4 Q96NZ8 WAP, kazal,immunoglobulin, kunitz and NTR domain-containing WFIKKN1 protein 1Q96NZ9 Proline-rich acidic protein 1 PRAP1 Q96P44 Collagen alpha-1(XXI)chain COL21A1 Q96PB7 Noelin-3 OLFM3 Q96PC5 Melanoma inhibitory activityprotein 2 MIA2 Q96PD5 N-acetylmuramoyl-L-alanine amidase PGLYRP2 Q96PH6Beta-defensin 118 DEFB118 Q96PL1 Secretoglobin family 3A member 2SCGB3A2 Q96PL2 Beta-tectorin TECTB Q96QH8 Sperm acrosome-associatedprotein 5 SPACA5 Q96QR1 Secretoglobin family 3A member 1 SCGB3A1 Q96QU1Protocadherin-15 PCDH15 Q96QV1 Hedgehog-interacting protein HHIP Q96RW7Hemicentin-1 HMCN1 Q96S42 Nodal homolog NODAL Q96S86 Hyaluronan andproteoglycan link protein 3 HAPLN3 Q96SL4 Glutathione peroxidase 7 GPX7Q96SM3 Probable carboxypeptidase X1 CPXM1 Q96T91 Glycoprotein hormonealpha-2 GPHA2 Q99062 Granulocyte colony-stimulating factor receptorCSF3R Q99102 Mucin-4 alpha chain MUC4 Q99217 Amelogenin, X isoform AMELXQ99218 Amelogenin, Y isoform AMELY Q99435 Protein kinase C-bindingprotein NELL2 NELL2 Q99470 Stromal cell-derived factor 2 SDF2 Q99542Matrix metalloproteinase-19 MMP19 Q99574 Neuroserpin SERPINI1 Q99584Protein S100-A13 S100A13 Q99616 C—C motif chemokine 13 CCL13 Q99645Epiphycan EPYC Q99674 Cell growth regulator with EF hand domain protein1 CGREF1 Q99715 Collagen alpha-1(XII) chain COL12A1 Q99727Metalloproteinase inhibitor 4 TIMP4 Q99731 C—C motif chemokine 19 CCL19Q99748 Neurturin NRTN Q99935 Proline-rich protein 1 PROL1 Q99942 E3ubiquitin-protein ligase RNF5 RNF5 Q99944 Epidermal growth factor-likeprotein 8 EGFL8 Q99954 Submaxillary gland androgen-regulated protein 3ASMR3A Q99969 Retinoic acid receptor responder protein 2 RARRES2 Q99972Myocilin MYOC Q99983 Osteomodulin OMD Q99985 Semaphorin-3C SEMA3C Q99988Growth/differentiation factor 15 GDF15 Q9BPW4 Apolipoprotein L4 APOL4Q9BQ08 Resistin-like beta RETNLB Q9BQ16 Testican-3 SPOCK3 Q9BQ51Programmed cell death 1 ligand 2 PDCD1LG2 Q9BQB4 Sclerostin SOST Q9BQI4Coiled-coil domain-containing protein 3 CCDC3 Q9BQP9 BPI fold-containingfamily A member 3 BPIFA3 Q9BQR3 Serine protease 27 PRSS27 Q9BQY6 WAPfour-disulfide core domain protein 6 WFDC6 Q9BRR6 ADP-dependentglucokinase ADPGK Q9BS86 Zona pellucida-binding protein 1 ZPBP Q9BSG0Protease-associated domain-containing protein 1 PRADC1 Q9BSG5 RetbindinRTBDN Q9BT30 Probable alpha-ketoglutarate-dependent dioxygenase ABH7ALKBH7 Q9BT56 Spexin C12orf39 Q9BT67 NEDD4 family-interacting protein 1NDFIP1 Q9BTY2 Plasma alpha-L-fucosidase FUCA2 Q9BU40 Chordin-likeprotein 1 CHRDL1 Q9BUD6 Spondin-2 SPON2 Q9BUN1 Protein MENT MENT Q9BUR5Apolipoprotein O APOO Q9BV94 ER degradation-enhancingalpha-mannosidase-like 2 EDEM2 Q9BWP8 Collectin-11 COLEC11 Q9BWS9Chitinase domain-containing protein 1 CHID1 Q9BX67 Junctional adhesionmolecule C JAM3 Q9BX93 Group XIIB secretory phospholipase A2-likeprotein PLA2G12B Q9BXI9 Complement C1q tumor necrosis factor-relatedprotein 6 C1QTNF6 Q9BXJ0 Complement C1q tumor necrosis factor-relatedprotein 5 C1QTNF5 Q9BXJ1 Complement C1q tumor necrosis factor-relatedprotein 1 C1QTNF1 Q9BXJ2 Complement C1q tumor necrosis factor-relatedprotein 7 C1QTNF7 Q9BXJ3 Complement C1q tumor necrosis factor-relatedprotein 4 C1QTNF4 Q9BXJ4 Complement C1q tumor necrosis factor-relatedprotein 3 C1QTNF3 Q9BXJ5 Complement C1q tumor necrosis factor-relatedprotein 2 C1QTNF2 Q9BXN1 Asporin ASPN Q9BXP8 Pappalysin-2 PAPPA2 Q9BXR6Complement factor H-related protein 5 CFHR5 Q9BXS0 Collagen alpha-1(XXV)chain COL25A1 Q9BXX0 EMILIN-2 EMILIN2 Q9BXY4 R-spondin-3 RSPO3 Q9BY15EGF-like module-containing mucin-like hormone receptor-like 3 EMR3subunit beta Q9BY50 Signal peptidase complex catalytic subunit SEC11CSEC11C Q9BY76 Angiopoietin-related protein 4 ANGPTL4 Q9BYF1 Processedangiotensin-converting enzyme 2 ACE2 Q9BYJ0 Fibroblast growthfactor-binding protein 2 FGFBP2 Q9BYW3 Beta-defensin 126 DEFB126 Q9BYX4Interferon-induced helicase C domain-containing protein 1 IFIH1 Q9BYZ8Regenerating islet-derived protein 4 REG4 Q9BZ76 Contactin-associatedprotein-like 3 CNTNAP3 Q9BZG9 Ly-6/neurotoxin-like protein 1 LYNX1Q9BZJ3 Tryptase delta TPSD1 Q9BZM1 Group XIIA secretory phospholipase A2PLA2G12A Q9BZM2 Group IIF secretory phospholipase A2 PLA2G2F Q9BZM5NKG2D ligand 2 ULBP2 Q9BZP6 Acidic mammalian chitinase CHIA Q9BZZ2Sialoadhesin SIGLEC1 Q9C0B6 Protein FAM5B FAM5B Q9GZM7Tubulointerstitial nephritis antigen-like TINAGL1 Q9GZN4 Brain-specificserine protease 4 PRSS22 Q9GZP0 Platelet-derived growth factor D,receptor-binding form PDGFD Q9GZT5 Protein Wnt-10a WNT10A Q9GZU5Nyctalopin NYX Q9GZV7 Hyaluronan and proteoglycan link protein 2 HAPLN2Q9GZV9 Fibroblast growth factor 23 FGF23 Q9GZX9 Twisted gastrulationprotein homolog 1 TWSG1 Q9GZZ7 GDNF family receptor alpha-4 GFRA4 Q9GZZ8Extracellular glycoprotein lacritin LACRT Q9H0B8 Cysteine-rich secretoryprotein LCCL domain-containing 2 CRISPLD2 Q9H106 Signal-regulatoryprotein delta SIRPD Q9H114 Cystatin-like 1 CSTL1 Q9H173 Nucleotideexchange factor SIL1 SIL1 Q9H1E1 Ribonuclease 7 RNASE7 Q9H1F0 WAPfour-disulfide core domain protein 10A WFDC10A Q9H1J5 Protein Wnt-8aWNT8A Q9H1J7 Protein Wnt-5b WNT5B Q9H1M3 Beta-defensin 129 DEFB129Q9H1M4 Beta-defensin 127 DEFB127 Q9H1Z8 Augurin C2orf40 Q9H239 Matrixmetalloproteinase-28 MMP28 Q9H2A7 C—X—C motif chemokine 16 CXCL16 Q9H2A9Carbohydrate sulfotransferase 8 CHST8 Q9H2R5 Kallikrein-15 KLK15 Q9H2X0Chordin CHRD Q9H2X3 C-type lectin domain family 4 member M CLEC4M Q9H306Matrix metalloproteinase-27 MMP27 Q9H324 A disintegrin andmetalloproteinase with thrombospondin motifs 10 ADAMTS10 Q9H336Cysteine-rich secretory protein LCCL domain-containing 1 CRISPLD1 Q9H3E2Sorting nexin-25 SNX25 Q9H3R2 Mucin-13 MUC13 Q9H3U7 SPARC-relatedmodular calcium-binding protein 2 SMOC2 Q9H3Y0 Peptidase inhibitorR3HDML R3HDML Q9H4A4 Aminopeptidase B RNPEP Q9H4F8 SPARC-related modularcalcium-binding protein 1 SMOC1 Q9H4G1 Cystatin-9-like CST9L Q9H5V8 CUBdomain-containing protein 1 CDCP1 Q9H6B9 Epoxide hydrolase 3 EPHX3Q9H6E4 Coiled-coil domain-containing protein 134 CCDC134 Q9H741 UPF0454protein C12orf49 C12orf49 Q9H772 Gremlin-2 GREM2 Q9H7Y0 Deleted inautism-related protein 1 CXorf36 Q9H8L6 Multimerin-2 MMRN2 Q9H9S5Fukutin-related protein FKRP Q9HAT2 Sialate O-acetylesterase SIAE Q9HB40Retinoid-inducible serine carboxypeptidase SCPEP1 Q9HB63 Netrin-4 NTN4Q9HBJ0 Placenta-specific protein 1 PLAC1 Q9HC23 Prokineticin-2 PROK2Q9HC57 WAP four-disulfide core domain protein 1 WFDC1 Q9HC73 Cytokinereceptor-like factor 2 CRLF2 Q9HC84 Mucin-5B MUC5B Q9HCB6 Spondin-1SPON1 Q9HCQ7 Neuropeptide NPSF NPVF Q9HCT0 Fibroblast growth factor 22FGF22 Q9HD89 Resistin RETN Q9NNX1 Tuftelin TUFT1 Q9NNX6 CD209 antigenCD209 Q9NP55 BPI fold-containing family A member 1 BPIFA1 Q9NP70Ameloblastin AMBN Q9NP95 Fibroblast growth factor 20 FGF20 Q9NP99Triggering receptor expressed on myeloid cells 1 TREM1 Q9NPA2 Matrixmetalloproteinase-25 MMP25 Q9NPE2 Neugrin NGRN Q9NPH0 Lysophosphatidicacid phosphatase type 6 ACP6 Q9NPH6 Odorant-binding protein 2b OBP2BQ9NQ30 Endothelial cell-specific molecule 1 ESM1 Q9NQ36 Signal peptide,CUB and EGF-like domain-containing protein 2 SCUBE2 Q9NQ38 Serineprotease inhibitor Kazal-type 5 SPINK5 Q9NQ76 Matrix extracellularphosphoglycoprotein MEPE Q9NQ79 Cartilage acidic protein 1 CRTAC1 Q9NR16Scavenger receptor cysteine-rich type 1 protein M160 CD163L1 Q9NR23Growth/differentiation factor 3 GDF3 Q9NR71 Neutral ceramidase ASAH2Q9NR99 Matrix-remodeling-associated protein 5 MXRA5 Q9NRA1Platelet-derived growth factor C PDGFC Q9NRC9 Otoraplin OTOR Q9NRE1Matrix metalloproteinase-26 MMP26 Q9NRJ3 C—C motif chemokine 28 CCL28Q9NRM1 Enamelin ENAM Q9NRN5 Olfactomedin-like protein 3 OLFML3 Q9NRR1Cytokine-like protein 1 CYTL1 Q9NS15 Latent-transforming growth factorbeta-binding protein 3 LTBP3 Q9NS62 Thrombospondin type-1domain-containing protein 1 THSD1 Q9NS71 Gastrokine-1 GKN1 Q9NS98Semaphorin-3G SEMA3G Q9NSA1 Fibroblast growth factor 21 FGF21 Q9NT22EMILIN-3 EMILIN3 Q9NTU7 Cerebellin-4 CBLN4 Q9NVR0 Kelch-like protein 11KLHL11 Q9NWH7 Spermatogenesis-associated protein 6 SPATA6 Q9NXC2Glucose-fructose oxidoreductase domain-containing protein 1 GFOD1 Q9NY56Odorant-binding protein 2a OBP2A Q9NY84 Vascular non-inflammatorymolecule 3 VNN3 Q9NZ20 Group 3 secretory phospholipase A2 PLA2G3 Q9NZC2Triggering receptor expressed on myeloid cells 2 TREM2 Q9NZK5 Adenosinedeaminase CECR1 CECR1 Q9NZK7 Group IIE secretory phospholipase A2PLA2G2E Q9NZP8 Complement C1r subcomponent-like protein C1RL Q9NZV1Cysteine-rich motor neuron 1 protein CRIM1 Q9NZW4 Dentin sialoproteinDSPP Q9P0G3 Kallikrein-14 KLK14 Q9P0W0 Interferon kappa IFNK Q9P218Collagen alpha-1(XX) chain COL20A1 Q9P2C4 Transmembrane protein 181TMEM181 Q9P2K2 Thioredoxin domain-containing protein 16 TXNDC16 Q9P2N4 Adisintegrin and metalloproteinase with thrombospondin motifs 9 ADAMTS9Q9UBC7 Galanin-like peptide GALP Q9UBD3 Cytokine SCM-1 beta XCL2 Q9UBD9Cardiotrophin-like cytokine factor 1 CLCF1 Q9UBM4 Opticin OPTC Q9UBP4Dickkopf-related protein 3 DKK3 Q9UBQ6 Exostosin-like 2 EXTL2 Q9UBR5Chemokine-like factor CKLF Q9UBS5 Gamma-aminobutyric acid type Breceptor subunit 1 GABBR1 Q9UBT3 Dickkopf-related protein 4 short formDKK4 Q9UBU2 Dickkopf-related protein 2 DKK2 Q9UBU3 Ghrelin-28 GHRLQ9UBV4 Protein Wnt-16 WNT16 Q9UBX5 Fibulin-5 FBLN5 Q9UBX7 Kallikrein-11KLK11 Q9UEF7 Klotho KL Q9UFP1 Protein FAM198A FAM198A Q9UGM3 Deleted inmalignant brain tumors 1 protein DMBT1 Q9UGM5 Fetuin-B FETUB Q9UGP8Translocation protein SEC63 homolog SEC63 Q9UHF0 Neurokinin-B TAC3Q9UHF1 Epidermal growth factor-like protein 7 EGFL7 Q9UHG2 ProSAASPCSK1N Q9UHI8 A disintegrin and metalloproteinase with thrombospondinmotifs 1 ADAMTS1 Q9UHL4 Dipeptidyl peptidase 2 DPP7 Q9UI42Carboxypeptidase A4 CPA4 Q9UIG4 Psoriasis susceptibility 1 candidategene 2 protein PSORS1C2 Q9UIK5 Tomoregulin-2 TMEFF2 Q9UIQ6Leucyl-cystinyl aminopeptidase, pregnancy serum form LNPEP Q9UJA9Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 ENPP5Q9UJH8 Meteorin METRN Q9UJJ9 N-acetylglucosamine-1-phosphotransferasesubunit gamma GNPTG Q9UJW2 Tubulointerstitial nephritis antigen TINAGQ9UK05 Growth/differentiation factor 2 GDF2 Q9UK55 Protein Z-dependentprotease inhibitor SERPINA10 Q9UK85 Dickkopf-like protein 1 DKKL1 Q9UKJ1Paired immunoglobulin-like type 2 receptor alpha PILRA Q9UKP4 Adisintegrin and metalloproteinase with thrombospondin motifs 7 ADAMTS7Q9UKP5 A disintegrin and metalloproteinase with thrombospondin motifs 6ADAMTS6 Q9UKQ2 Disintegrin and metalloproteinase domain-containingprotein 28 ADAM28 Q9UKQ9 Kallikrein-9 KLK9 Q9UKR0 Kallikrein-12 KLK12Q9UKR3 Kallikrein-13 KLK13 Q9UKU9 Angiopoietin-related protein 2 ANGPTL2Q9UKZ9 Procollagen C-endopeptidase enhancer 2 PCOLCE2 Q9UL52Transmembrane protease serine 11E non-catalytic chain TMPRSS11E Q9ULC0Endomucin EMCN Q9ULI3 Protein HEG homolog 1 HEG1 Q9ULZ1 Apelin-13 APLNQ9ULZ9 Matrix metalloproteinase-17 MMP17 Q9UM21Alpha-1,3-mannosyl-glycoprotein 4-beta-N- MGAT4Aacetylglucosaminyltransferase A soluble form Q9UM22 Mammalianependymin-related protein 1 EPDR1 Q9UM73 ALK tyrosine kinase receptorALK Q9UMD9 97 kDa linear IgA disease antigen COL17A1 Q9UMX5 NeudesinNENF Q9UN73 Protocadherin alpha-6 PCDHA6 Q9UNA0 A disintegrin andmetalloproteinase with thrombospondin motifs 5 ADAMTS5 Q9UNI1Chymotrypsin-like elastase family member 1 CELA1 Q9UNK4 Group IIDsecretory phospholipase A2 PLA2G2D Q9UP79 A disintegrin andmetalloproteinase with thrombospondin motifs 8 ADAMTS8 Q9UPZ6Thrombospondin type-1 domain-containing protein 7A THSD7A Q9UQ72Pregnancy-specific beta-1-glycoprotein 11 PSG11 Q9UQ74Pregnancy-specific beta-1-glycoprotein 8 PSG8 Q9UQC9 Calcium-activatedchloride channel regulator 2 CLCA2 Q9UQE7 Structural maintenance ofchromosomes protein 3 SMC3 Q9UQP3 Tenascin-N TNN Q9Y223UDP-N-acetylglucosamine 2-epimerase GNE Q9Y240 C-type lectin domainfamily 11 member A CLEC11A Q9Y251 Heparanase 8 kDa subunit HPSE Q9Y258C—C motif chemokine 26 CCL26 Q9Y264 Angiopoietin-4 ANGPT4 Q9Y275 Tumornecrosis factor ligand superfamily member 13b, membrane TNFSF13B formQ9Y287 BRI2 intracellular domain ITM2B Q9Y2E5 Epididymis-specificalpha-mannosidase MAN2B2 Q9Y334 von Willebrand factor Adomain-containing protein 7 VWA7 Q9Y337 Kallikrein-5 KLK5 Q9Y3B3Transmembrane emp24 domain-containing protein 7 TMED7 Q9Y3E2 BolA-likeprotein 1 BOLA1 Q9Y426 C2 domain-containing protein 2 C2CD2 Q9Y4K0 Lysyloxidase homolog 2 LOXL2 Q9Y4X3 C—C motif chemokine 27 CCL27 Q9Y5C1Angiopoietin-related protein 3 ANGPTL3 Q9Y5I2 Protocadherin alpha-10PCDHA10 Q9Y5I3 Protocadherin alpha-1 PCDHA1 Q9Y5K2 Kallikrein-4 KLK4Q9Y5L2 Hypoxia-inducible lipid droplet-associated protein HILPDA Q9Y5Q5Atrial natriuretic peptide-converting enzyme CORIN Q9Y5R2 Matrixmetalloproteinase-24 MMP24 Q9Y5U5 Tumor necrosis factor receptorsuperfamily member 18 TNFRSF18 Q9Y5W5 Wnt inhibitory factor 1 WIF1Q9Y5X9 Endothelial lipase LIPG Q9Y625 Secreted glypican-6 GPC6 Q9Y646Carboxypeptidase Q CPQ Q9Y6C2 EMILIN-1 EMILIN1 Q9Y6F9 Protein Wnt-6 WNT6Q9Y6I9 Testis-expressed sequence 264 protein TEX264 Q9Y6L7 Tolloid-likeprotein 2 TLL2 Q9Y6N3 Calcium-activated chloride channel regulatorfamily member 3 CLCA3P Q9Y6N6 Laminin subunit gamma-3 LAMC3 Q9Y6R7IgGFc-binding protein FCGBP Q9Y6Y9 Lymphocyte antigen 96 LY96 Q9Y6Z7Collectin-10 COLEC10

In some embodiments, the compositions and methods of the inventionprovide for the delivery of one or more mRNAs encoding one or moreadditional exemplary proteins listed in Table 2; thus, compositions ofthe invention may comprise an mRNA encoding a protein listed in Table 2(or a homolog thereof) along with other components set out herein, andmethods of the invention may comprise preparing and/or administering acomposition comprising an mRNA encoding a protein chosen from theproteins listed in Table 2 (or a homolog thereof) along with othercomponents set out herein.

TABLE 2 Additional Exemplary Proteins Uniprot ID Protein Name Gene NameA6NGW2 Putative stereocilin-like protein STRCP1 A6NIE9 Putative serineprotease 29 PRSS29P A6NJ16 Putative V-set and immunoglobulin IGHV4OR15-8domain-containing-like protein IGHV4OR15-8 A6NJS3 Putative V-set andimmunoglobulin IGHV1OR21-1 domain-containing-like protein IGHV1OR21-1A6NMY6 Putative annexin A2-like protein ANXA2P2 A8MT79 Putativezinc-alpha-2-glycoprotein-like 1 A8MWS1 Putative killer cellimmunoglobulin-like KIR3DP1 receptor like protein KIR3DP1 A8MXU0Putative beta-defensin 108A DEFB108P1 C9JUS6 Putativeadrenomedullin-5-like protein ADM5 P0C7V7 Putative signal peptidasecomplex SEC11B catalytic subunit SEC11B P0C854 Putative cat eye syndromecritical region CECR9 protein 9 Q13046 Putative pregnancy-specificbeta-1- PSG7 glycoprotein 7 Q16609 Putative apolipoprotein(a)-likeprotein 2 LPAL2 Q2TV78 Putative macrophage-stimulating protein MST1P9MSTP9 Q5JQD4 Putative peptide YY-3 PYY3 Q5R387 Putative inactive groupIIC secretory PLA2G2C phospholipase A2 Q5VSP4 Putative lipocalin 1-likeprotein 1 LCN1P1 Q5W188 Putative cystatin-9-like protein CST9LP1 CST9LP1Q6UXR4 Putative serpin A13 SERPINA13P Q86SH4 Putative testis-specificprion protein PRNT Q86YQ2 Putative latherin LATH Q8IVG9 Putative humaninpeptide MT-RNR2 Q8NHM4 Putative trypsin-6 TRY6 Q8NHW4 C-C motifchemokine 4-like CCL4L2 Q9H7L2 Putative killer cell immunoglobulin-likeKIR3DX1 receptor-like protein KIR3DX1 Q9NRI6 Putative peptide YY-2 PYY2Q9UF72 Putative TP73 antisense gene protein 1 TP73-AS1 Q9UKY3 Putativeinactive carboxylesterase 4 CES1P1

The Uniprot IDs set forth in Table 1 and Table 2 refer to the humanversions the listed proteins and the sequences of each are availablefrom the Uniprot database. Sequences of the listed proteins are alsogenerally available for various animals, including various mammals andanimals of veterinary or industrial interest. Accordingly, in someembodiments, compositions and methods of the invention provide for thedelivery of one or more mRNAs encoding one or more proteins chosen frommammalian homologs or homologs from an animal of veterinary orindustrial interest of the secreted proteins listed in Table 1 or Table2; thus, compositions of the invention may comprise an mRNA encoding aprotein chosen from mammalian homologs or homologs from an animal ofveterinary or industrial interest of a protein listed in Table 1 orTable 2 along with other components set out herein, and methods of theinvention may comprise preparing and/or administering a compositioncomprising an mRNA encoding a protein chosen from mammalian homologs orhomologs from an animal of veterinary or industrial interest of aprotein listed in Table 1 or Table 2 along with other components set outherein. In some embodiments, mammalian homologs are chosen from mouse,rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat,ferret, sheep, goat, or camel homologs. In some embodiments, the animalof veterinary or industrial interest is chosen from the mammals listedabove and/or chicken, duck, turkey, salmon, catfish, or tilapia.

In embodiments, the compositions and methods of the invention providefor the delivery of mRNA encoding a lysosomal protein chosen from Table3. In some embodiments, the compositions and methods of the inventionprovide for the delivery of one or more mRNAs encoding one or morelysosomal and/or related proteins listed in Table 3; thus, compositionsof the invention may comprise an mRNA encoding a protein listed in Table3 (or a homolog thereof) along with other components set out herein, andmethods of the invention may comprise preparing and/or administering acomposition comprising an mRNA encoding a protein chosen from theproteins listed in Table 3 (or a homolog thereof) along with othercomponents set out herein.

TABLE 3 Lysosomal and Related Proteins α-fucosidase α-galactosidaseα-glucosidase α-Iduronidase α-mannosidase α-N-acetylgalactosaminidase(α-galactosidase B) β-galactosidase β-glucuronidase β-hexosaminidaseβ-mannosidase 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase3-methylcrotonyl-CoA carboxylase 3-O-sulfogalactosyl cerebrosidesulfatase (arylsulfatase A) acetyl-CoA transferase acidalpha-glucosidase acid ceramidase acid lipase acid phosphatase acidsphingomyelinase alpha-galactosidase A arylsulfatase Abeta-galactosidase beta-glucocerebrosidase beta-hexosaminidasebiotinidase cathepsin A cathepsin K CLN3 CLN5 CLN6 CLN8 CLN9 cystinetransporter (cystinosin) cytosolic protein beta3A subunit of the adaptorprotein-3 complex, AP3 formyl-Glycine generating enzyme (FGE)galactocerebrosidase galactose-1-phosphate uridyltransferase (GALT)galactose 6-sulfate sulfatase (also known asN-acetylgalactosamine-6-sulfatase) glucocerebrosidase glucuronatesulfatase glucuronidase glycoprotein cleaving enzymes glycosaminoglycancleaving enzymes glycosylasparaginase (aspartylglucosaminidase) GM2-APHeparan-alpha-glucosaminide N-acetyltransferase (HGSNAT, TMEM76) Heparansulfatase hexosaminidase A lysosomal proteases methylmalonyl-CoA mutasehyaluronidase Iduronate sulfatase LAMP-2 lysosomal α-mannosidaseLysosomal p40 (C2orf18) Major facilitator superfamily domain containing8 protein (MFSD8 or CLN7) N-acetylgalactosamine 4-sulfatase N-acetylglucosamine 6-sulfatase N-acetyl glucosaminidaseN-acetylglucosamine-1-phosphate transferase NPC1 NPC2 palmitoyl-proteinthioesterase palmitoyl-protein thioesterase (CLN1) Saposin A(Sphingolipid activator protein A) Saposin B (Sphingolipid activatorprotein B) Saposin C (Sphingolipid activator protein C) Saposin D(Sphingolipid activator protein D) sialic acid transporter (sialin)sialidase Sialin sulfatase Transmembrane protein 74 (TMEM74)tripeptidyl-peptidase tripeptidyl-peptidase I (CLN2)UDP-N-acetylglucosamine-phosphotransferase

Information regarding lysosomal proteins is available from Lubke et al.,“Proteomics of the Lysosome,” Biochim Biophys Acta. (2009) 1793:625-635. In some embodiments, the protein listed in Table 3 and encodedby mRNA in the compositions and methods of the invention is a humanprotein. Sequences of the listed proteins are also available for variousanimals, including various mammals and animals of veterinary orindustrial interest as described above.

In some embodiments, the compositions and methods of the inventionprovide for the delivery of mRNA encoding a therapeutic protein (e.g.,cytosolic, transmembrane or secreted) such as those listed in Table 4.In some embodiments, the compositions and methods of the inventionprovide for the delivery of an mRNA encoding a therapeutic proteinuseful in treating a disease or disorder (i.e., indication) listed inTable 4; thus, compositions of the invention may comprise an mRNAencoding a therapeutic protein listed or not listed in Table 4 (or ahomolog thereof, as discussed below) along with other components set outherein for treating a disease or disorder (i.e., indication) listed inTable 4, and methods of the invention may comprise preparing and/oradministering a composition comprising an mRNA encoding a such a protein(or a homolog thereof, as discussed below) along with other componentsset out herein for treatment of a disease or disorder listed in Table 4.

TABLE 4 Exemplary Indications and Related Proteins IndicationTherapeutic Protein 3-Methylcrotonyl-CoA carboxylase Methylcrotonoyl-CoAdeficiency carboxylase 3-Methylglutaconic aciduria Methylglutaconyl-CoAhydratase Actinic keratosis Acute intermittent porphyria Porphobilinogendeaminase Acute lymphocytic leukemia Acute myeloid leukemia Addison'sdisease Adenosine deaminase deficiency Adenosine deaminaseAdrenoleukodystrophy ABCD1 Adrenomyeloneuropathy AIDS/HIV Alcohol usedisorders Alkaptonuria Homogentisate 1,2-dioxygenase Allergic asthmaAnti-IgE mAb Allergies (dermatitis, rhinitis) Alopecia areata Alpers'disease POLG Alpers-Huttenlocher syndrome Alpha 1-antitrypsin deficiencyAlpha 1 protease inhibitor Alpha-mannosidosis Alpha-D-mannosidase Alportsyndrome Alzheimer's disease Amyloid light-chain amyloidosis Amyotrophiclateral sclerosis (ALS) Anemia Erythropoietin Aortic valve stenosisArgininemia Arginase Argininosuccinic acidemia Argininosuccinate lyaseArrhythmogenic right ventricular dysplasia Autism Autosomal dominant andrecessive pro- gressive external ophthalmoplegia with mitochondrial DNAdeletions Autosomal recessive polycystic ARPKD kidney disease Bacterialinfections Basal cell carcinoma Batten disease Battenin + others B-cellchronic lymphocytic leukemia Becker muscular dystrophy DystrophinBeta-thalassemia Beta globin Binge eating disorder Bipolar disorderBladder cancer Blepharospasm, Cervical dystonia, Botulinum toxin Chronicmigraine, more Bronchiolitis obliterans Brugada syndrome Buerger'sdisease CACNA1A CACNB4-related Episodic Ataxia Type 2 Cancer anddepression Cancer and sexual dysfunction Cancer in pregnancyCarbamylphosphate synthetase deficiency Carbamylphosphate synthetaseCarcinoma of the gallbladder Cardiomyopathy (diabetic) Cardiomyopathy(hypertrophic) Carnitine uptake defect SLC22A5 Catecholaminergicpolymorphic ventricular tachycardia CDKL5-related Atypical Rett SyndromeCeliac disease Cellulitis Cerebrovascular disease Cervix uteri cancerChronic fatigue syndrome Chronic graft versus host disease Chronicidiopathic urticaria Chronic immune thrombocytopenia ThrombopoietinChronic kidney kisease Chronic liver disease Chronic lymphocyticleukemia Chronic myeloid leukemia Chronic pancreatitis Cirrhosis of theliver Citrullinemia, type I Argininosuccinate synthase Classic RettSyndrome Classical galactosemia Galactose-1-phosphateuridylyltransferase Clostridium difficile associated diarrhea Clottingdisorders COAD/COPD Cocaine addiction COL4A5-related disorders Coldcontact urticaria Contraception, female Coronary artery diseases Corpusuteri cancer Corticobasal degeneration Crigler-Najjar syndromeUDP-glucuronosyl- transferase Critical limb ischemia CTNS-relatedcystinosis Cutaneous lupus erythematosus Cutaneous neuroendocrinecarcinoma (Merkel Cell) Cystic fibrosis CFTR Cystic fibrosisDeoxyribonuclease I Cystinosis Cystinosin Cystinuria SLC7A9 Dementia(Lewy body) Depression Diabetic foot infections Diabetic foot ulcerDiabetic peripheral neuropathy Diabetic ulcers Diarrhoeal diseasesDiffuse large B-cell lymphoma DiGeorge syndrome Diverticulitis Drug usedisorders Duchenne muscular dystrophy Dystrophin Dysarthria Dyskinesia(levodopa-induced) Early-onset autosomal dominant Alzheimer's diseaseEczema Ehlers-Danlos syndrome, type 1 EIF2B1 EIF2B2 EIF2B3 EIF2B4EIF2B5-related childhood ataxia with central nervous system hypomyeli-nation/vanishing white matter Eosinophilic esophagitis Epilepsy Erectiledysfunction Erythropoietic protoporphyria Ferrochelatase Esophagealcarcinoma Essential tremor Fabry disease Alpha galactosidase Familialadenomatous polyposis APC Familial chylomicronemia Lipoprotein lipaseFamilial dysbetalipoproteinemia Apolipoprotein E Familial isolateddilated cardiomyopathy Familial mediterranean fever Pyrin (MEFV)Familial melanoma Female infertility Follicle stimulating hormone Femalesexual dysfunction Fibromyalgia FMR1-related disorders Fracture healingFragile X Premature Ovarian Failure Syndrome Fragile X syndrome FMRPFragile X-Associated Tremor/Ataxia Syndrome Friedreich's ataxiaFrontotemporal dementia Fryns syndrome Galactocerebrosidase deficienciesGALE deficiency Galactose epimerase GALK deficiency GalactokinaseGALT-related galactosemia Gastric cancer Gastroesophageal reflux diseaseGaucher disease Glucocerebrosidase Gilbert syndrome UDP-glucuronosyl-transferase Glioblastoma multiforme Glomerulonephritis Glutaricacidemia, type I Glutaryl-CoA dehydrogenase GM2 gangliosidosis HEXA,HEXB Gout Urate oxidase Graft versus host disease Growth hormonedeficiency Growth hormone 1/ Growth hormone 2 Head and neck cancer,Metastatic Anti-EGFr mAb colorectal cancer Hearing loss, adult onsetHeart failure Hemachromatosis HFE protein Hemifacial spasm Hemolyticuremic syndrome Anti-complement factor C5 mAb Hemophilia A Factor VIIIHemophilia A, Hemophilia B Factor VII Hemophilia B Factor IX HepatitisB, Hepatitis C Interferon alpha HER2+ breast cancer, gastric cancerAnti-HER2 mAb Hereditary angioedema C1 esterase inhibitor Hereditaryhemorrhagic telangiectasia Hereditary hemorrhagic telangiectasia (AT)Hereditary spherocytosis Hidradenitis suppurativa HomocystinuriaCystathionine beta-synthase Homozygous familial hypercholesterolemia LDLreceptor Hunter syndrome (MPS II) Iduronate-2-sulfatase Huntingtondisease Huntingtin Hurler syndrome (MPS I) Alpha-L iduronidaseHydrolethalus Hyperalgesia Hyperbilirubinemia HyperhidrosisHyperlipidemia Hypermethioninemia Methionine adenosyl- transferaseHyperoxaluria, type I Serine-pyruvate amino- transferase HypertensionHyperuricemia Hyponatremia Hypoparathyroidism Parathyroid hormoneHypophosphatasia TNSALP Idiopathic pulmonary fibrosis IminoglycinuriaImmunoglobulin deficiency Immunoglobulin Infection (adenovirus)Infection (anthrax prophylaxis) Infection (BK virus) Infection(Clostridium difficile prophylaxis) Infection (Dengue fever prophylaxis)Infection (Epstein-Barr virus) Infection (Hepatitis-D) Infection (Lymedisease prophylaxis) Infection (Smallpox virus) Infectious diseasesvaccines Infectious antigen Inflammatory heart diseases InsomniaInterstitial cystitis Iron-deficiency anaemia Irritable bowel diseaseIschaemic heart disease Isovaleric aciduria Isovaleric acid CoAdehydrogenase deficiency Jansky-Bielschowsky disease Juvenile Battendisease Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) Juvenilerheumatoid arthritis TNF-alpha inhibitors Kennedy's disease (SBMA)Keratoconus Krabbe disease Galactocerebrosidase Leber's hereditary opticneuropathy NADH dehydrogenase Leiomyosarcoma Lennox-Gastaut syndromeLesch-Nyhan syndrome Hypoxanthine phos- phoribosyltransferase 1Leukaemia Li-Fraumeni syndrome TP53 Lipoma Liposarcoma Liver cancerLong-chain 3-OH acyl-CoA Long-chain-3-hydroxyacyl- dehydrogenasedeficiency CoA dehydrogenase Lower respiratory infections Lysosomal acidlipase deficiency Lysosomal acid lipase Macular degeneration Majordepressive disorder Malignant fibrous histiocytoma Mantle cell lymphomaMaple syrup urine disease 3-methyl-2-oxobutanoate dehydrogenase Marfansyndrome FBN1 Maroteaux-Lamy syndrome (MPS VI) N-acetylgalactosamine4-sulfatase Mastocytosis McArdle disease Muscle glycogen phosphorylaseMECP2-related disorders MECP2-related Severe Neonatal EncephalopathyMedium-chain acyl-CoA dehydrogenase Acyl-CoA dehydrogenase deficiencyMelanoma Anti-CTLA4 mAb Metachromatic leukodystrophy Arylsulfatase AMetastatic colorectal cancer, NSCLC, others Anti-VEGF mAbMethylmalonyl-CoA mutase deficiency Methylmalonyl-CoA mutase MigraineMitochondrial oxidative phosphorylation disorders Morquio syndrome, typeA (MPS IVA) Galactose 6-sulfate sulfatase Morquio syndrome, type B (MPSIVB) Beta-galactosidase Mouth and oropharynx cancers Multiplecarboxylase deficiency Biotin-methylcrotonoyl- CoA-carboxylase ligaseMultiple myeloma Multiple sclerosis Anti-VLA-4 mAb Multiple sclerosisInterferon beta Multiple system atrophy Myasthenia gravis MyelofibrosisNarcolepsy Neonatal bronchopulmonary dysplasia Neonatal infectionsNephritis and nephrosis Neurofibromatosis, type 1 NF-1 Neuronal ceroidlipofuscinoses- related diseases Neutropenia G-CSF Niemann Pick disease,type A/B SMPD1 Niemann Pick disease, type C NPC1 Niemann-Pick diseaseType C1 Nocturia Non-alcoholic fatty liver disease Non-Hodgkin lymphomaAnti-CD20 mAb Non-small cell lung cancer Notch-3 related cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL) Obesity Ophthalmoparesis Opioid inducedconstipation Ornithine transcarbamylase deficiency Ornithinetranscarbamylase Osteoarthritis Osteopetrosis Osteoporosis Anti-RANKLmAb Ovarian cancer Paget disease of bone Sequestosome 1 Pain Pancreaticcarcinoma Panic disorder Parkinson disease Paroxysmal nocturnalhemoglobinuria Anti-complement factor C5 Mab Pediculosis capitis (headlice) Pelizaeus-Merzbacher disease Pemphigus vulgaris Peptic ulcerdisease Peripheral neuropathy Peyronie's disease PhenylketonuriaPhenylalanine hydroxylase Pneumococcal infection prophylaxisPOLG-related sensory ataxic neuropathy Polycystic kidney diseasePolycystic ovary syndrome Polycythaemia vera Polymerase G-relateddisorders Polymorphous light eruption Pompe disease Alpha glucosidasePorphyria cutanea tarda Uroporphyrinogen decarboxylase Post herpeticneuralgia Post-organ transplant Pouchitis PPM-X Syndrome Prader-Willisyndrome Preeclampsia Premature ejaculation Prematurity and low birthweight Primary ciliary dyskinesia Primary glomerular diseases Primaryhumoral immune Immunoglobulin deficiencies (e.g., CVID) ProctitisProgressive multifocal leukoencephalopathy Progressive supranuclearpalsy Propionic acidemia Propionyl-CoA carboxylase Prostate cancerPsoriasis Anti-IL-12 & IL-23 mAb Psoriatic arthritis TNF-alphainhibitors PTT-1 Pulmonary arterial hypertension Pulmonary arterialhypertension Raynaud's phenomenon Refractive errors Renal cell carcinomaRestless leg syndrome Retinitis pigmentosa Rheumatic heart diseaseRheumatoid arthritis Anti-interleukin-6 (IL-6) mAb Rheumatoid arthritisT-cell costimulation blocker Rheumatoid arthritis TNF-alpha inhibitorRomano-Ward syndrome Rosacea Sanfilippo syndrome, type A (MPS IIIA)Heparan N-sulfatase Sanfilippo syndrome, type B (MPS IIIB)N-acetyl-alpha-D- glucosaminidase Santavuori-Haltia diseaseSchizophrenia Schnitzler syndrome Scleroderma SCN1A SCN1B-relatedseizure disorders Short-chain acyl-CoA dehydrogenase Butyryl-CoAdeficiency dehydrogenase Sickle cell disease Hemoglobin SLC3A1-relateddisorders Small cell lung cancer SMN-1-related spinal muscular atrophy(SMA) Spinal muscular atrophy Survival motor neuron protein Squamouscell carcinoma of head and neck Stickler syndrome Stomach cancer Strokeprophylaxis Synovial sarcoma Systemic lupus erythematosus Anti-BAFFSystemic sclerosis Tetrahydrobiopterin-deficient Tetrahydrobiopterinhyperphenylalaninemia Thromboangiitis obliterans Thrombotic disordersThyroid cancer TPP1 deficiencies Trachea, bronchus, lung cancersTricuspid atresia TSC1 TSC2-related tuberous sclerosis Type 2 diabetesmellitus Glucagon-like peptide 1 (GLP-1) agonist Type 2 diabetesmellitus Insulin Tyrosinemia, type I Fumarylacetoacetase Ulcerativecolitis Uterine fibroids Varicose veins Venous thromboembolism Verylong-chain acyl-CoA dehydro- Long-chain-acyl-CoA genase deficiencydehydrogenase von Gierke's disease Glucose-6-phosphatase VonHippel-Lindau disease pVHL Wegener granulomatosis Wilson disease Wilsondisease protein X-Linked adrenal hypoplasia X-linkedadrenoleukodystrophy X-linked agammaglobulinemia Bruton's tyrosinekinase

In some embodiments, the present invention is used to prevent, treatand/or cure a subject affected with a disease or disorder listed orassociated with the proteins listed in Tables 1, 2, 3 or 4. In someembodiments, an mRNA encodes one or more of Cystic FibrosisTransmembrane Conductance Regulator (CFTR), argininosuccinate synthetase(ASS1), Factor IX, survival motor neuron 1 (SMN1), or phenylalaninehydroxylase (PAH).

In some embodiments, an mRNA encoding any one of the proteins listed inTables 1, 2, 3 or 4 is codon-optimized. In one embodiment, an mRNAencoding CFTR is codon-optimized.

TABLE 5 Human CFTR Codon-AUGCAACGCUCUCCUCUUGAAAAGGCCUCGGUGGUGUCCAAGCUCUU OptimizedCUUCUCGUGGACUAGACCCAUCCUGAGAAAGGGGUACAGACAGCGCU HumanUGGAGCUGUCCGAUAUCUAUCAAAUCCCUUCCGUGGACUCCGCGGAC CFTRAACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUC mRNAAAAGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUC codingUGGCGGUUCAUGUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCAC sequenceCAAGGCCGUGCAGCCCCUGUUGCUGGGACGGAUUAUUGCCUCCUACGACCCCGACAACAAGGAAGAAAGAAGCAUCGCUAUCUACUUGGGCAUCGGUCUGUGCCUGCUUUUCAUCGUCCGGACCCUCUUGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAUGAGAAUUGCCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCUUGACAAGAUUUCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUCUGAACAAGUUCGACGAGGGCCUCGCCCUGGCCCACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGCUUCUGAUGGGCCUGAUCUGGGAGCUGCUGCAAGCCUCGGCAUUCUGUGGGCUUGGAUUCCUGAUCGUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAUGGAAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCCGCAAGGCCGCUUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUCCGGGUUCUUCGUGGUGUUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAGGGAAUCAUCCUCAGGAAGAUCUUCACCACCAUUUCCUUCUGUAUCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCCAUGGGCCGUGCAGACUUGGUACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGACUUCCUUCAAAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACUACUACCGAGGUCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUGUUCGAGAAGGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCCCUCUUCUUUUCAAACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACAUUAACUUCAAGAUCGAAAGAGGACAGCUCCUGGCGGUGGCCGGAUCGACCGGAGCCGGAAAGACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAACCUAGCGAGGGAAAGAUCAAGCACUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCGGAACCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUACCGCUACCGGUCCGUGAUCAAAGCCUGCCAGCUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGAUAACAUCGUGCUGGGCGAAGGGGGUAUUACCUUGUCGGGGGGCCAGCGGGCUAGAAUCUCGCUGGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGGACUCCCCCUUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGUGCAAGCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGCACCUGAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCCUCCUACUUUUACGGCACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUUCUCAUCGAAGCUGAUGGGUUGCGACAGCUUCGACCAGUUCUCCGCCGAAAGAAGGAACUCGAUCCUGACGGAAACCUUGCACCGCUUCUCUUUGGAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAAGAAGCAGAGCUUCAAGCAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAUCUUGAACCCCAUUAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCCACUGCAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCGCCUGUCCCUGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGUGAUCUCCACUGGUCCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUGAACCUGAUGACCCACAGCGUGAACCAGGGCCAAAACAUUCACCGCAAGACUACCGCAUCCACCCGGAAAGUGUCCCUGGCACCUCAAGCGAAUCUUACCGAGCUCGACAUCUACUCCCGGAGACUGUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAGGAUCUGAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGUGACGACUUGGAACACUUAUCUGCGGUACAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUGGUGCCUGGUGAUUUUCCUGGCCGAGGUCGCGGCCUCACUGGUGGUGCUCUGGCUGUUGGGAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUCGAGAAACAACAGCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGUCGGAGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGCUGGUCCACACCUUGAUCACCGUCAGCAAGAUUCUUCACCACAAGAUGUUGCAUAGCGUGCUGCAGGCCCCCAUGUCCACCCUCAACACUCUGAAGGCCGGAGGCAUUCUGAACAGAUUCUCCAAGGACAUCGCUAUCCUGGACGAUCUCCUGCCGCUUACCAUCUUUGACUUCAUCCAGCUGCUGCUGAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUGCAGCCUUACAUUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGCGGGCCUACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGAGGGACGAUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGACUGUGGACCCUCCGGGCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGGCCCUGAACCUCCACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCGGUGGUUCCAGAUGCGCAUCGAGAUGAUUUUCGUCAUCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACUACCGGAGAGGGAGAGGGACGGGUCGGAAUAAUCCUGACCCUCGCCAUGAACAUUAUGAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACAGCCUGAUGCGAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCACUAAGUCCACUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAAAACUCCCACGUGAAGAAGGACGAUAUUUGGCCCUCCGGAGGUCAAAUGACCGUGAAGGACCUGACCGCAAAGUACACCGAGGGAGGAAACGCCAUUCUCGAAAACAUCAGCUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUCCGGGAAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGGGGAAAUCCAAAUUGACGGCGUGUCUUGGGAUUCCAUUACUCUGCAGCAGUGGCGGAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCUCGGGUACCUUCCGGAAGAACCUGGAUCCUUACGAGCAGUGGAGCGACCAAGAAAUCUGGAAGGUCGCCGACGAGGUCGGCCUGCGCUCCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUCGACGGGGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGUCCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCCCACCUGGAUCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCAGGCCUUUGCCGAUUGCACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUGCUGGAGUGCCAGCAGUUCCUGGUCAUCGAGGAGAACAAGGUCCGCCAAUACGACUCCAUUCAAAAGCUCCUCAACGAGCGGUCGCUGUUCAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUCCCGCAUCGGAACAGCUCAAAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGGAAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAA (SEQ ID NO: 1) ComparisonAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUU Codon-CUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGC OptimizedUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAU HumanAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUC CFTRUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCU mRNAGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCAC codingAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACG sequenceACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA (SEQ ID NO: 2) Codon-AUGCAGCGGUCCUAGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUU OptimizedCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGC HumanUUGAGUUGUCUGACAUCUACCAGUGACCCUCGGUAGAUUCGGCGGAU CFTRAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUC ‘STOP’UAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCU CodingGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCAC SequenceAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA (SEQ ID NO: 3) HumanMQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEK CFTRLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLL ProteinGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIA SequenceMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 4)Exemplary Codon-Optimized Human Cystic Fibrosis TransmembraneConductance Regulator (CFTR) mRNAs

Construct design: X - Coding Sequence - Y 5′ and 3′ UTR Sequences: X (5′UTR Sequence) = (SEQ ID NO: 5)GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG Y (3′ UTR Sequence) =(SEQ ID NO: 6) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC AAGCU  OR(SEQ ID NO: 7) GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCA AAGCU

In one embodiment, a codon-optimized human CFTR mRNA sequence includesSEQ ID NO: 1.

In one embodiment, a full-length codon-optimized human CFTR mRNAsequence is:

(SEQ ID NO: 8) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAACGCUCUCCUCUUGAAAAGGCCUCGGUGGUGUCCAAGCUCUUCUUCUCGUGGACUAGACCCAUCCUGAGAAAGGGGUACAGACAGCGCUUGGAGCUGUCCGAUAUCUAUCAAAUCCCUUCCGUGGACUCCGCGGACAACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUCAAAGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUCUGGCGGUUCAUGUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCACCAAGGCCGUGCAGCCCCUGUUGCUGGGACGGAUUAUUGCCUCCUACGACCCCGACAACAAGGAAGAAAGAAGCAUCGCUAUCUACUUGGGCAUCGGUCUGUGCCUGCUUUUCAUCGUCCGGACCCUCUUGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAUGAGAAUUGCCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCUUGACAAGAUUUCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUCUGAACAAGUUCGACGAGGGCCUCGCCCUGGCCCACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGCUUCUGAUGGGCCUGAUCUGGGAGCUGCUGCAAGCCUCGGCAUUCUGUGGGCUUGGAUUCCUGAUCGUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAUGGAAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCCGCAAGGCCGCUUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUCCGGGUUCUUCGUGGUGUUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAGGGAAUCAUCCUCAGGAAGAUCUUCACCACCAUUUCCUUCUGUAUCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCCAUGGGCCGUGCAGACUUGGUACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGACUUCCUUCAAAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACUACUACCGAGGUCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUGUUCGAGAAGGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCCCUCUUCUUUUCAAACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACAUUAACUUCAAGAUCGAAAGAGGACAGCUCCUGGCGGUGGCCGGAUCGACCGGAGCCGGAAAGACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAACCUAGCGAGGGAAAGAUCAAGCACUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCGGAACCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUACCGCUACCGGUCCGUGAUCAAAGCCUGCCAGCUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGAUAACAUCGUGCUGGGCGAAGGGGGUAUUACCUUGUCGGGGGGCCAGCGGGCUAGAAUCUCGCUGGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGGACUCCCCCUUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGUGCAAGCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGCACCUGAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCCUCCUACUUUUACGGCACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUUCUCAUCGAAGCUGAUGGGUUGCGACAGCUUCGACCAGUUCUCCGCCGAAAGAAGGAACUCGAUCCUGACGGAAACCUUGCACCGCUUCUCUUUGGAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAAGAAGCAGAGCUUCAAGCAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAUCUUGAACCCCAUUAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCCACUGCAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCGCCUGUCCCUGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGUGAUCUCCACUGGUCCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUGAACCUGAUGACCCACAGCGUGAACCAGGGCCAAAACAUUCACCGCAAGACUACCGCAUCCACCCGGAAAGUGUCCCUGGCACCUCAAGCGAAUCUUACCGAGCUCGACAUCUACUCCCGGAGACUGUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAGGAUCUGAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGUGACGACUUGGAACACUUAUCUGCGGUACAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUGGUGCCUGGUGAUUUUCCUGGCCGAGGUCGCGGCCUCACUGGUGGUGCUCUGGCUGUUGGGAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUCGAGAAACAACAGCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGUCGGAGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGCUGGUCCACACCUUGAUCACCGUCAGCAAGAUUCUUCACCACAAGAUGUUGCAUAGCGUGCUGCAGGCCCCCAUGUCCACCCUCAACACUCUGAAGGCCGGAGGCAUUCUGAACAGAUUCUCCAAGGACAUCGCUAUCCUGGACGAUCUCCUGCCGCUUACCAUCUUUGACUUCAUCCAGCUGCUGCUGAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUGCAGCCUUACAUUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGCGGGCCUACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGAGGGACGAUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGACUGUGGACCCUCCGGGCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGGCCCUGAACCUCCACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCGGUGGUUCCAGAUGCGCAUCGAGAUGAUUUUCGUCAUCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACUACCGGAGAGGGAGAGGGACGGGUCGGAAUAAUCCUGACCCUCGCCAUGAACAUUAUGAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACAGCCUGAUGCGAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCACUAAGUCCACUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAAAACUCCCACGUGAAGAAGGACGAUAUUUGGCCCUCCGGAGGUCAAAUGACCGUGAAGGACCUGACCGCAAAGUACACCGAGGGAGGAAACGCCAUUCUCGAAAACAUCAGCUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUCCGGGAAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGGGGAAAUCCAAAUUGACGGCGUGUCUUGGGAUUCCAUUACUCUGCAGCAGUGGCGGAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCUCGGGUACCUUCCGGAAGAACCUGGAUCCUUACGAGCAGUGGAGCGACCAAGAAAUCUGGAAGGUCGCCGACGAGGUCGGCCUGCGCUCCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUCGACGGGGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGUCCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCCCACCUGGAUCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCAGGCCUUUGCCGAUUGCACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUGCUGGAGUGCCAGCAGUUCCUGGUCAUCGAGGAGAACAAGGUCCGCCAAUACGACUCCAUUCAAAAGCUCCUCAACGAGCGGUCGCUGUUCAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUCCCGCAUCGGAACAGCUCAAAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGGAAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

In one embodiment, another full-length codon-optimized human CFTR mRNAsequence is:

(SEQ ID NO: 9) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAACGCUCUCCUCUUGAAAAGGCCUCGGUGGUGUCCAAGCUCUUCUUCUCGUGGACUAGACCCAUCCUGAGAAAGGGGUACAGACAGCGCUUGGAGCUGUCCGAUAUCUAUCAAAUCCCUUCCGUGGACUCCGCGGACAACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACAGAGAACUCGCCUCAAAGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUCUGGCGGUUCAUGUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCACCAAGGCCGUGCAGCCCCUGUUGCUGGGACGGAUUAUUGCCUCCUACGACCCCGACAACAAGGAAGAAAGAAGCAUCGCUAUCUACUUGGGCAUCGGUCUGUGCCUGCUUUUCAUCGUCCGGACCCUCUUGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAUGAGAAUUGCCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCUUGACAAGAUUUCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUCUGAACAAGUUCGACGAGGGCCUCGCCCUGGCCCACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGCUUCUGAUGGGCCUGAUCUGGGAGCUGCUGCAAGCCUCGGCAUUCUGUGGGCUUGGAUUCCUGAUCGUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGAAAGAUUUCCGAACGGCUGGUGAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAUGGAAAAGAUGAUUGAAAACCUCCGGCAAACCGAGCUGAAGCUGACCCGCAAGGCCGCUUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUCCGGGUUCUUCGUGGUGUUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAGGGAAUCAUCCUCAGGAAGAUCUUCACCACCAUUUCCUUCUGUAUCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCCAUGGGCCGUGCAGACUUGGUACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGACUUCCUUCAAAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACUACUACCGAGGUCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUGUUCGAGAAGGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCCCUCUUCUUUUCAAACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACAUUAACUUCAAGAUCGAAAGAGGACAGCUCCUGGCGGUGGCCGGAUCGACCGGAGCCGGAAAGACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAACCUAGCGAGGGAAAGAUCAAGCACUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCGGAACCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUACCGCUACCGGUCCGUGAUCAAAGCCUGCCAGCUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGAUAACAUCGUGCUGGGCGAAGGGGGUAUUACCUUGUCGGGGGGCCAGCGGGCUAGAAUCUCGCUGGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGGACUCCCCCUUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGUGCAAGCUGAUGGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGCACCUGAAGAAGGCAGACAAGAUUCUGAUUCUGCAUGAGGGGUCCUCCUACUUUUACGGCACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUUCUCAUCGAAGCUGAUGGGUUGCGACAGCUUCGACCAGUUCUCCGCCGAAAGAAGGAACUCGAUCCUGACGGAAACCUUGCACCGCUUCUCUUUGGAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAAGAAGCAGAGCUUCAAGCAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAUCUUGAACCCCAUUAACUCCAUCCGCAAGUUCUCAAUCGUGCAAAAGACGCCACUGCAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCGCCUGUCCCUGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGUGAUCUCCACUGGUCCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUGAACCUGAUGACCCACAGCGUGAACCAGGGCCAAAACAUUCACCGCAAGACUACCGCAUCCACCCGGAAAGUGUCCCUGGCACCUCAAGCGAAUCUUACCGAGCUCGACAUCUACUCCCGGAGACUGUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAGGAUCUGAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGUGACGACUUGGAACACUUAUCUGCGGUACAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUGGUGCCUGGUGAUUUUCCUGGCCGAGGUCGCGGCCUCACUGGUGGUGCUCUGGCUGUUGGGAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUCGAGAAACAACAGCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGUCGGAGUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGCUGGUCCACACCUUGAUCACCGUCAGCAAGAUUCUUCACCACAAGAUGUUGCAUAGCGUGCUGCAGGCCCCCAUGUCCACCCUCAACACUCUGAAGGCCGGAGGCAUUCUGAACAGAUUCUCCAAGGACAUCGCUAUCCUGGACGAUCUCCUGCCGCUUACCAUCUUUGACUUCAUCCAGCUGCUGCUGAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUGCAGCCUUACAUUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGCGGGCCUACUUCCUCCAAACCAGCCAGCAGCUGAAGCAACUGGAAUCCGAGGGACGAUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGACUGUGGACCCUCCGGGCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGGCCCUGAACCUCCACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCGGUGGUUCCAGAUGCGCAUCGAGAUGAUUUUCGUCAUCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACUACCGGAGAGGGAGAGGGACGGGUCGGAAUAAUCCUGACCCUCGCCAUGAACAUUAUGAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACAGCCUGAUGCGAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCACUAAGUCCACUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAAAACUCCCACGUGAAGAAGGACGAUAUUUGGCCCUCCGGAGGUCAAAUGACCGUGAAGGACCUGACCGCAAAGUACACCGAGGGAGGAAACGCCAUUCUCGAAAACAUCAGCUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUCCGGGAAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGGGGAAAUCCAAAUUGACGGCGUGUCUUGGGAUUCCAUUACUCUGCAGCAGUGGCGGAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCUCGGGUACCUUCCGGAAGAACCUGGAUCCUUACGAGCAGUGGAGCGACCAAGAAAUCUGGAAGGUCGCCGACGAGGUCGGCCUGCGCUCCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUCGACGGGGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGUCCGUGCUCUCCAAGGCCAAGAUUCUGCUGCUGGACGAACCUUCGGCCCACCUGGAUCCGGUCACCUACCAGAUCAUCAGGAGGACCCUGAAGCAGGCCUUUGCCGAUUGCACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUGCUGGAGUGCCAGCAGUUCCUGGUCAUCGAGGAGAACAAGGUCCGCCAAUACGACUCCAUUCAAAAGCUCCUCAACGAGCGGUCGCUGUUCAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUCCCGCAUCGGAACAGCUCAAAGUGCAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGGAAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU

In one embodiment, a codon-optimized human CFTR mRNA sequence includesSEQ ID NO: 2.

In one embodiment, a full-length codon-optimized human CFTR mRNAsequence is:

(SEQ ID NO: 10) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAA GCU

In one embodiment, another full-length codon-optimized human CFTR mRNAsequence is:

(SEQ ID NO: 11) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAA GCU

SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 include 5′and 3′ untranslated regions framing a codon-optimized hCFTR-encodingmRNA.

In some embodiments, a suitable mRNA sequence may be an mRNA sequence ahomolog or an analog of human CFTR (hCFTR) protein. For example, ahomolog or an analog of hCFTR protein may be a modified hCFTR proteincontaining one or more amino acid substitutions, deletions, and/orinsertions as compared to a wild-type or naturally-occurring hCFTRprotein while retaining substantial hCFTR protein activity. In someembodiments, an mRNA suitable for the present invention encodes an aminoacid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO:4. In some embodiments, an mRNA suitable for the present inventionencodes a protein substantially identical to hCFTR protein. In someembodiments, an mRNA suitable for the present invention encodes an aminoacid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:4. In some embodiments, an mRNA suitable for the present inventionencodes a fragment or a portion of hCFTR protein. In some embodiments,an mRNA suitable for the present invention encodes a fragment or aportion of hCFTR protein, wherein the fragment or portion of the proteinstill maintains CFTR activity similar to that of the wild-type protein.In some embodiments, an mRNA suitable for the present invention has anucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical SEQID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, an mRNAsuitable for the present invention has a nucleotide sequence at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical SEQ ID NO: 2, SEQ ID NO: 10 or SEQID NO: 11. In some embodiments, an mRNA suitable for the presentinvention comprises a nucleotide sequence identical to SEQ ID NO: 1. Insome embodiments, an mRNA suitable for the present invention comprises anucleotide sequence identical to SEQ ID NO: 8. In some embodiments, anmRNA suitable for the present invention comprises a nucleotide sequenceidentical to SEQ ID NO: 9. In some embodiments, a suitable mRNA encodesa fusion protein comprising a full length, fragment or portion of anhCFTR protein fused to another protein (e.g., an N or C terminalfusion). In some embodiments, the protein fused to the mRNA encoding afull length, fragment or portion of an hCFTR protein encodes a signal ora cellular targeting sequence.

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

Non-Coding Regions

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA moleculeswhich are stable (e.g., globin, actin, GAPDH, tubulin, histone, orcitric acid cycle enzymes) to increase the stability of the sense mRNAmolecule. For example, a 5′ UTR sequence may include a partial sequenceof a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improvethe nuclease resistance and/or improve the half-life of thepolynucleotide. Also contemplated is the inclusion of a sequenceencoding human growth hormone (hGH), or a fragment thereof to the 3′ endor untranslated region of the polynucleotide (e.g., mRNA) to furtherstabilize the polynucleotide. Generally, these modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of thepolynucleotide relative to their unmodified counterparts, and include,for example modifications made to improve such polynucleotides'resistance to in vivo nuclease digestion.

According to various embodiments, any size mRNA may be encapsulated byprovided liposomes. In some embodiments, the provided liposomes mayencapsulate mRNA of greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., mRNAs encoding CFTR) include a5′ cap structure. A 5′ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups fromthe 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. Additional cap structures aredescribed in published US Application No. US 2016/0032356 and U.S.Provisional Application 62/464,327, filed Feb. 27, 2017, which areincorporated herein by reference.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a2′-O-methyl residue of the ribose attached to bases 1 and 2. In someembodiments, the cap is a Cap1 structure. Cap1 structures have a2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2structure. Cap2 structures have a 2′-O-methyl residue attached to bothbases 2 and 3.

In some embodiments, mRNAs (e.g., mRNAs encoding CFTR) include a 3′ tailstructure. Typically, a tail structure includes a poly(A) and/or poly(C)tail. A poly-A or poly-C tail on the 3′ terminus of mRNA typicallyincludes at least 50 adenosine or cytosine nucleotides, at least 150adenosine or cytosine nucleotides, at least 200 adenosine or cytosinenucleotides, at least 250 adenosine or cytosine nucleotides, at least300 adenosine or cytosine nucleotides, at least 350 adenosine orcytosine nucleotides, at least 400 adenosine or cytosine nucleotides, atleast 450 adenosine or cytosine nucleotides, at least 500 adenosine orcytosine nucleotides, at least 550 adenosine or cytosine nucleotides, atleast 600 adenosine or cytosine nucleotides, at least 650 adenosine orcytosine nucleotides, at least 700 adenosine or cytosine nucleotides, atleast 750 adenosine or cytosine nucleotides, at least 800 adenosine orcytosine nucleotides, at least 850 adenosine or cytosine nucleotides, atleast 900 adenosine or cytosine nucleotides, at least 950 adenosine orcytosine nucleotides, or at least 1 kb adenosine or cytosinenucleotides, respectively. In some embodiments, a poly-A or poly-C tailmay be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine orcytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides,about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosinenucleotides, about 50 to 600 adenosine or cytosine nucleotides, about100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosineor cytosine nucleotides, about 200 to 600 adenosine or cytosinenucleotides, about 250 to 600 adenosine or cytosine nucleotides, about300 to 600 adenosine or cytosine nucleotides, about 350 to 600 adenosineor cytosine nucleotides, about 400 to 600 adenosine or cytosinenucleotides, about 450 to 600 adenosine or cytosine nucleotides, about500 to 600 adenosine or cytosine nucleotides, about 10 to 150 adenosineor cytosine nucleotides, about 10 to 100 adenosine or cytosinenucleotides, about 20 to 70 adenosine or cytosine nucleotides, or about20 to 60 adenosine or cytosine nucleotides) respectively. In someembodiments, a tail structure includes is a combination of poly(A) andpoly(C) tails with various lengths described herein. In someembodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%,80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosinenucleotides. In some embodiments, a tail structure includes at least50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or99% cytosine nucleotides.

Modified mRNA

In some embodiments, mRNA according to the present invention may besynthesized as unmodified or modified mRNA. Typically, mRNAs aremodified to enhance stability. Modifications of mRNA can include, forexample, modifications of the nucleotides of the RNA. A modified mRNAaccording to the invention can thus include, for example, backbonemodifications, sugar modifications or base modifications. In someembodiments, mRNAs may be synthesized from naturally occurringnucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purines (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g., from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732,4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418,5,153,319, 5,262,530 and 5,700,642, the disclosures of which areincorporated by reference in their entirety.

In some embodiments, mRNAs (e.g., mRNAs encoding CFTR) may contain RNAbackbone modifications. Typically, a backbone modification is amodification in which the phosphates of the backbone of the nucleotidescontained in the RNA are modified chemically. Exemplary backbonemodifications typically include, but are not limited to, modificationsfrom the group consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g., cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., mRNAs encoding CFTR) may contain sugarmodifications. A typical sugar modification is a chemical modificationof the sugar of the nucleotides it contains including, but not limitedto, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), azidotriphosphates(2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate) or 4′-thio-substituted ribonucleotides.

In some embodiments, mRNAs (e.g., mRNAs encoding CFTR) may containmodifications of the bases of the nucleotides (base modifications). Amodified nucleotide which contains a base modification is also called abase-modified nucleotide. Examples of such base-modified nucleotidesinclude, but are not limited to, 2-amino-6-chloropurine riboside5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine5′-triphosphate, O6-methylguanosine 5′-triphosphate, pseudouridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

Pharmaceutical Compositions

To facilitate expression of mRNA in vivo, delivery vehicles such asliposomes can be formulated in combination with one or more additionalnucleic acids, carriers, targeting ligands or stabilizing reagents, orin pharmacological compositions where it is mixed with suitableexcipients. Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

Provided liposomally-encapsulated or associated mRNAs, and compositionscontaining the same, may be administered and dosed in accordance withcurrent medical practice, taking into account the clinical condition ofthe subject, the site and method of administration, the scheduling ofadministration, the subject's age, sex, body weight and other factorsrelevant to clinicians of ordinary skill in the art. The “effectiveamount” for the purposes herein may be determined by such relevantconsiderations as are known to those of ordinary skill in experimentalclinical research, pharmacological, clinical and medical arts. In someembodiments, the amount administered is effective to achieve at leastsome stabilization, improvement or elimination of symptoms and otherindicators as are selected as appropriate measures of disease progress,regression or improvement by those of skill in the art. For example, asuitable amount and dosing regimen is one that causes at least transientprotein (e.g., enzyme) production.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, and/or intranasal administration.

Alternately or additionally, liposomally encapsulated mRNAs andcompositions of the invention may be administered in a local rather thansystemic manner, for example, via injection of the pharmaceuticalcomposition directly into a targeted tissue, preferably in a sustainedrelease formulation. Local delivery can be affected in various ways,depending on the tissue to be targeted. For example, aerosols containingcompositions of the present invention can be inhaled (for nasal,tracheal, or bronchial delivery); compositions of the present inventioncan be injected into the site of injury, disease manifestation, or pain,for example; compositions can be provided in lozenges for oral,tracheal, or esophageal application; can be supplied in liquid, tabletor capsule form for administration to the stomach or intestines, can besupplied in suppository form for rectal or vaginal application; or caneven be delivered to the eye by use of creams, drops, or even injection.Formulations containing provided compositions complexed with therapeuticmolecules or ligands can even be surgically administered, for example inassociation with a polymer or other structure or substance that canallow the compositions to diffuse from the site of implantation tosurrounding cells. Alternatively, they can be applied surgically withoutthe use of polymers or supports.

In some embodiments, provided liposomes and/or compositions areformulated such that they are suitable for extended-release of the mRNAcontained therein. Such extended-release compositions may beconveniently administered to a subject at extended dosing intervals. Forexample, in one embodiment, the compositions of the present inventionare administered to a subject twice day, daily or every other day. In apreferred embodiment, the compositions of the present invention areadministered to a subject twice a week, once a week, every ten days,every two weeks, every three weeks, or more preferably every four weeks,once a month, every six weeks, every eight weeks, every other month,every three months, every four months, every six months, every eightmonths, every nine months or annually. Also contemplated arecompositions and liposomes which are formulated for depot administration(e.g., intramuscularly, subcutaneously, intravitreally) to eitherdeliver or release mRNA over extended periods of time. Preferably, theextended-release means employed are combined with modifications made tothe mRNA to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomes disclosed herein and relatedmethods for the use of such compositions as disclosed for example, inU.S. Provisional Application No. 61/494,882, filed Jun. 8, 2011, theteachings of which are incorporated herein by reference in theirentirety. For example, lyophilized pharmaceutical compositions accordingto the invention may be reconstituted prior to administration or can bereconstituted in vivo. For example, a lyophilized pharmaceuticalcomposition can be formulated in an appropriate dosage form (e.g., anintradermal dosage form such as a disk, rod or membrane) andadministered such that the dosage form is rehydrated over time in vivoby the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desiredtissue. In some embodiments, the mRNA delivered by provided liposomes orcompositions is expressed in the tissue in which the liposomes and/orcompositions were administered. In some embodiments, the mRNA deliveredis expressed in a tissue different from the tissue in which theliposomes and/or compositions were administered. Exemplary tissues inwhich delivered mRNA may be delivered and/or expressed include, but arenot limited to the lungs, liver, kidney, heart, spleen, serum, brain,skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.

In some embodiments, a target tissue is lung. In some embodiments, atarget tissue is the upper (i.e., superior) lobe of the right or leftlung. In some embodiments, a target tissue is the lower (i.e., inferior)lobe of the right or left lung. In some embodiments, a target tissue isthe middle lobe of the right lung.

In some embodiments, a target tissue is the apical segment of the rightlung or the apicoposterior segment of the left lung. In someembodiments, a target tissue is the posterior segment of the right lung.In some embodiments, a target tissue is the anterior segment of theright or left lung. In some embodiments, a target tissue is the superiorsegment of the right or left lung. In some embodiments, a target tissueis the lateral basal segment of the right or left lung. In someembodiments, a target tissue is the anterior basal segment of the rightlung. In some embodiments, a target tissue is the anteromedial basalsegment of the left lung. In some embodiments, a target tissue is thelateral segment of the right lung. In some embodiments, a target tissueis the medial segment of the right lung. In some embodiments, a targettissue is the superior lingular segment of the left lung. In someembodiments, a target tissue is the inferior lingular segment of theleft lung. In some embodiments, a target tissue is the posterior basalsegment of the right or left lung. In some embodiments, a target tissueis the medial basal segment of the right lung.

In particular embodiments, a target tissue is epithelial cells in thelung. In some embodiments, a target tissue is smooth muscle cells in thelung. In some embodiment, a target tissue is pancreatic duct epithelialcells. In some embodiment, a target tissue is bile-duct epithelialcells. In some embodiment, a target tissue is epithelial cells of thesalivary glands. In some embodiment, a target tissue is renal epithelialcells. In some embodiment, a target tissue is beta-S cells in sweatgland secretory coils of sweat glands. In some embodiment, a targettissue is epithelial cells of the reproductive tract.

According to various embodiments, the timing of expression of deliveredmRNAs can be tuned to suit a particular medical need. In someembodiments, the expression of the protein encoded by delivered mRNA isdetectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72hours in serum or target tissues after a single administration ofprovided liposomes or compositions. In some embodiments, the expressionof the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days,4 days, 5 days, 6 days, and/or 7 days in serum or target tissues after asingle administration of provided liposomes or compositions. In someembodiments, the expression of the protein encoded by the mRNA isdetectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks in serum or targettissues after a single administration of provided liposomes orcompositions. In some embodiments, the expression of the protein encodedby the mRNA is detectable after a month or longer after a singleadministration of provided liposomes or compositions.

In some embodiments, mRNA (e.g., encoding CFTR protein) in a formulationas provided herein (e.g. encapsulated in a lipid nanoparticle consistingof 3 distinct lipid components, one of which is a sterol-based cationiclipid) delivered to the lung (e.g., by nebulization) is expressed inlung tissue for at least 7 days, at least 14 days, at least 21 days, orat least 28 days. In some embodiments, mRNA (e.g., encoding CFTRprotein) in a formulation as provided herein (e.g. encapsulated in alipid nanoparticle consisting of 3 distinct lipid components, one ofwhich is a sterol-based cationic lipid) delivered to the lung (e.g., bynebulization) is expressed in lung tissue for up to 7 days, up to 14days, up to 21 days, or up to 28 days. In some embodiments, a protein(e.g., CFTR) encoded by an mRNA (e.g., encoding CFTR protein) in aformulation as provided herein (e.g. encapsulated in a lipidnanoparticle consisting of 3 distinct lipid components, one of which isa sterol-based cationic lipid) delivered to the lung (e.g., bynebulization) is expressed in lung tissue for at least 7 days, at least14 days, at least 21 days, or at least 28 days. In some embodiments, aprotein (e.g., CFTR) encoded by an mRNA (e.g., encoding CFTR protein) ina formulation as provided herein (e.g. encapsulated in a lipidnanoparticle consisting of 3 distinct lipid components, one of which isa sterol-based cationic lipid) delivered to the lung (e.g., bynebulization) is expressed in lung tissue for up to 7 days, up to 14days, up to 21 days, or up to 28 days.

The present invention can be used to deliver mRNA at various doses. Insome embodiments, an mRNA is administered at a dose ranging from about0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0, 0.1-3.5,0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3, 0.3-5.0,0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0,0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0,0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, an mRNA isadministered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0,2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg bodyweight.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate theinvention and are not intended to limit the same.

Lipid Materials

The formulations described herein included a multi-component lipidmixture of varying ratios employing cationic lipids, helper lipids andPEGy-modified lipids designed to encapsulate various nucleic acid-basedmaterials. Cationic lipids can include (but not exclusively)sterol-based cationic lipids, ICE (imidazole cholesterol ester),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-Dioleyloxy-3-dimethylaminopropane (DODMA),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), C12-200,DLinSSDMA, Target 24, etc. Helper lipids can include (but notexclusively) DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.The PEG-modified lipids can include (but not exclusively) apoly(ethylene) glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C₆-C₂₀ length.

N/P ratio is the molar ratios of the nitrogen (amine) groups of cationiccarriers to those of the phosphate ones of mRNA.

Messenger RNA Material

The formulations described herein included messenger RNA (mRNA). mRNAcan (but not exclusively) encode human Cystic Fibrosis TransmembraneConductance Regulator (CFTR) and Firefly Luciferase (FFL). ExemplarymRNA coding sequences as disclosed herein include SEQ ID NO: 1, SEQ IDNO: 2, and SEQ ID NO: 3. An exemplary 5′ UTR mRNA sequence as disclosedherein is SEQ ID NO: 5. Exemplary 3′ UTR mRNA sequences as disclosedherein are SEQ ID NO: 6 and SEQ ID NO: 7. Exemplary full length mRNAsequences (i.e., a coding sequence plus a 5′ UTR and a 3′ UTR) asdisclosed herein include SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, andSEQ ID NO: 11.

Exemplary LNP Formulations Comprising Different Cationic Lipids

In some embodiments, one particular application of the novel lipidcomposition comprising a sterol-based cationic lipid, a helper lipid,and a PEG or PEG-modified lipid is pulmonary delivery of mRNA, such asCFTR mRNA. Lipid nanoparticle formulations composed of ICE lipid, DOPElipid and DMG-PEG lipid (molar ratio of 60:35:5) exhibit high percentencapsulation values for mRNA, such as CFTR mRNA (>80%), as determinedby fluorescence-based detection of mRNA. As shown in Table 5, thisparticular formulation composition is superior because conventional pHtitrable cationic lipids with DOPE and DMG-PEG 2K do not show such highpercent encapsulation.

TABLE 5 The encapsulation percentage of mRNA for a sterol-based cationiclipid nanoparticle formulation and other cationic lipid-basednanoparticle formulations. All lipid formulations were prepared with thecomposition of cationic lipid:DOPE:DMG- PEG 2K (molar ratio 60:35:5). pHtitratable Cationic Lipid % mRNA encapsulation ICE 90 DODMA 51 DODAP 49C12-200 39 DLinSSDMA 57 Target 24 59

All of these exemplary formulations were prepared as 5 mg mRNA LNPpreparations.

Formulation Example #1 (ICE Lipid)

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 15 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 60 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der wall forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentration:0.5 mg/mL CFTR mRNA (encapsulated). % mRNA Encapsulation: 90%. Size: 60nm. PDI: 0.14.

Formulation Example #2 (DODMA Lipid)

Aliquots of 10 mg/mL ethanolic solutions of DODMA, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 15 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 60 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der wall forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentration:0.5 mg/mL CFTR mRNA (encapsulated). % mRNA Encapsulation: 51%. Size: 62nm. PDI: 0.18.

Formulation Example #3 (DODAP Lipid)

Aliquots of 10 mg/mL ethanolic solutions of DODAP, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 15 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 60 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der wall forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentration:0.5 mg/mL CFTR mRNA (encapsulated). % mRNA Encapsulation: 49%. Size: 78nm. PDI: 0.19.

Formulation Example #4 (C12-200 Lipid)

Aliquots of 10 mg/mL ethanolic solutions of C12-200, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 15 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 60 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der wall forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentration:0.5 mg/mL CFTR mRNA (encapsulated). % mRNA Encapsulation: 39%. Size: 98nm. PDI: 0.22.

Formulation Example #5 (DLin-SS-DMA Lipid)

Aliquots of 10 mg/mL ethanolic solutions of DLin-SS-DMA, DOPE andDMG-PEG2K (molar ratio of 60:35:5) were mixed and diluted with ethanolto 15 mL final volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 60 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der wall forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentration:0.5 mg/mL CFTR mRNA (encapsulated). % mRNA Encapsulation: 57%.

Formulation Example #6 (Target 24 Lipid)

Aliquots of 10 mg/mL ethanolic solutions of Target 24(3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione),DOPE and DMG-PEG2K (molar ratio of 60:35:5) were mixed and diluted withethanol to 15 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1mg/mL stock solution so as to have a final volume of 60 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der wall forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentration:0.5 mg/mL CFTR mRNA (encapsulated). % mRNA Encapsulation: 59%. Size: 92nm. PDI: 0.24.

Exemplary ICE Formulation Protocols A. Formulation Example—1 mg Scale,N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE, cholesterol andDMG-PEG2K (molar ratio of 60:35:5) were mixed and diluted with ethanolto 3 mL final volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution. The lipid solution was injected rapidly into the aqueousmRNA solution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated,concentrated and stored frozen until further use. Final concentration ofthe formulation was determined at 0.5 mg/mL CFTR mRNA (encapsulated).The formulation had about 89% mRNA encapsulation (89% of the lipidnanoparticles contained mRNA) and the lipid nanoparticles had an averagesize of around 67 nm with polydispersibility index (PDI) of 0.19.

B. Formulation Example—15 mg Scale, N/P=2

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 45 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 180 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles,which was a self-assembly process driven by electrostatic attraction andvan der Waals forces. The resultant formulation mixture was in citratebuffer containing 20% ethanol. The formulation was subjected to a bufferexchange and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR mRNA (encapsulated).The lipid nanoparticles had an average size of around 70 nm with PDI of0.17.

C. Formulation Example—15 mg Scale, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 45 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 180 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles.The resultant formulation mixture was in citrate buffer containing 20%ethanol. The formulation was subjected to a buffer exchange and theresultant formulation was adjusted to the desired mRNA concentration andstored frozen until further use. Final concentration of the formulationwas determined at 0.5 mg/mL CFTR mRNA (encapsulated). The formulationhad about 85% mRNA encapsulation and the lipid nanoparticles had anaverage size of around 65 nm with PDI of 0.13.

D. Formulation Example—30 mg Scale, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 90 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 360 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles.The resultant formulation mixture was in citrate buffer containing 20%ethanol. The formulation was subjected to a buffer exchange using TFFsystem and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR mRNA (encapsulated).The formulation had about 86% of mRNA encapsulation and the lipidnanoparticles had an average size of around 89 nm with PDI of 0.12.

E. Formulation Example—30 mg Scale, 3% PEG, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:37:3) were mixed and diluted with ethanol to 90 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 360 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles.The resultant formulation mixture was in citrate buffer containing 20%ethanol. The formulation was subjected to a buffer exchange using TFFsystem and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR mRNA (encapsulated).The lipid nanoparticles had an average size of around 94 nm with PDI of0.15.

F. Formulation Example—60 mg Scale, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 180 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 720 mL.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles.The resultant formulation mixture was in citrate buffer containing 20%ethanol. The formulation was subjected to a buffer exchange using TFFsystem and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR STOP mRNA(encapsulated). The formulation had about 97% mRNA encapsulation and thelipid nanoparticles had an average size of around 67 nm with PDI of0.12.

G. Formulation Example—3.5 Gram Scale, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 42 Lfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of CFTR mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 42 L.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles.The resultant formulation mixture was in citrate buffer containing 20%ethanol. The formulation was subjected to a buffer exchange using TFFsystem and the resultant formulation is adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR mRNA (encapsulated).The formulation had about 91% mRNA encapsulation and the lipidnanoparticles had an average size of around 50 nm with PDI of 0.17.

H. Formulation Example—200 mg Scale, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 600 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of FFL mRNA was prepared from a 1 mg/mLstock solution so as to have a final volume of 2.4 L.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams results in formation of lipid nanoparticles.The resultant formulation mixture is in citrate buffer containing 20%ethanol. The formulation is subjected to a buffer exchange using TFFsystem and the resultant formulation was adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR mRNA (encapsulated).The formulation had about 95% mRNA encapsulation and the lipidnanoparticles had an average size of around 59 nm with PDI of 0.15.

I. Formulation Example—200 mg Scale, N/P=4

Aliquots of 10 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K(molar ratio of 60:35:5) were mixed and diluted with ethanol to 600 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of hCFTR-STOP mRNA was prepared from a 1mg/mL stock solution so as to have a final volume of 2.4 L.

In this process, the lipids dissolved in ethanol and the mRNA dissolvedin citrate buffer were mixed using a pump system. The instantaneousmixing of the two streams resulted in formation of lipid nanoparticles.The resultant formulation mixture was in citrate buffer containing 20%ethanol. The formulation was subjected to a buffer exchange using TFFsystem and the resultant formulation is adjusted to the desired mRNAconcentration and stored frozen until further use. Final concentrationof the formulation was determined at 0.5 mg/mL CFTR STOP mRNA(encapsulated). The formulation had about 88% mRNA encapsulation and thelipid nanoparticles had an average size of around 57 nm with PDI of0.18.

Pulmonary Delivery of ICE LNPs

Several exemplary studies were performed to demonstrate successful mRNAdelivery and subsequent human CFTR protein production from ICE-basedLNPs encapsulating codon-optimized hCFTR mRNA. Immunohistochemicalanalysis was performed on all lung sections using an anti-human CFTRantibody to specifically detect human CFTR protein within the lungs oftreated animals.

Example 1. In Vivo Expression of hCFTR in Rat Lungs after IntratrachealAdministration

FIG. 1 shows exemplary immunohistochemical detection of hCFTR protein inmice lungs 24 hours after pulmonary delivery of hCFTR mRNA lipidnanoparticles prepared by the process described above (FormulationExample #1).

Mice were administered LNP formulations containing hCFTR mRNA viamicrosprayer (intratracheal aerosol). The LNP formulations were madeusing ICE lipid as the cationic lipid. The fixed lung tissues from thesemice were analyzed for the presence of hCFTR protein byimmunohistochemical staining.

Protein was detected throughout the entire lung including both thebronchial epithelial cells and the alveolar regions. Positive (brown)staining was observed in all mRNA lipid nanoparticle test articlegroups, as compared to the lack of brown staining in the lungs ofuntreated control mice. Panel A depicts untreated mouse lung at 10×magnification. Panel B depicts untreated mouse lung at 20×magnification. Panel C depicts codon-optimized hCFTR (CO-hCFTR) mRNA ICELNP-treated mouse lung at 10× magnification. Panel D depicts CO-hCFTRmRNA ICE LNP-treated mouse lung at 20× magnification.

Example 2. In Vivo Expression of hCFTR in Rat Lungs after NebulizationAdministration

FIG. 2 shows exemplary immunohistochemical detection of hCFTR protein inmice lungs 24 hours after pulmonary delivery of hCFTR mRNA lipidnanoparticles prepared by the process described above (FormulationExample #1).

Mice were administered, via nebulization, LNP formulations containinghCFTR mRNA. The LNP formulations were made using ICE lipid as thecationic lipid. The fixed lung tissues from these mice were analyzed forthe presence of hCFTR protein by immunohistochemical staining.

Protein was detected throughout the entire lung including both thebronchial epithelial cells and the alveolar regions. Positive (brown)staining was observed in all mRNA lipid nanoparticle test articlegroups, as compared to the lack of brown staining in the lungs ofuntreated control mice. Panel A depicts untreated mouse lung at 10×magnification. Panel B depicts untreated mouse lung at 20×magnification. Panel C depicts codon-optimized hCFTR (CO-hCFTR) mRNA ICELNP-treated mouse lung at 10× magnification. Panel D depicts CO-hCFTRmRNA ICE LNP-treated mouse lung at 20× magnification.

Example 3. In Vivo Expression of hCFTR in Rat Lungs (10 mg, 5% PEG)

FIG. 3 shows exemplary immunohistochemical analysis of hCFTR protein inrat lungs 24 hours after pulmonary delivery of codon-optimized hCFTR(CO-hCFTR) mRNA lipid nanoparticles prepared by the process describedabove using ICE-based lipid nanoparticles.

In some representative studies, rats were placed in an aerosol chamberwhich allowed for full motion of the rats. An aerosol was produced vianebulization of 10 mg (as measured by encapsulated mRNA) CO-hCFTR mRNAencapsulated in ICE LNPs (containing 5% [mol] of PEG-modified lipid)which filled the chamber containing the rats. The rats were exposed fora given period of time with during which the aerosol was freely takeninto the lungs via normal breathing. After the exposure, the rats wereremoved and placed back into their cages. The rats were then sacrificed24 hours after exposure and the lungs were harvested (a portion was snapfrozen and a separate portion was fixed in 10% NBF and embedded inparaffin) for analysis. The fixed lung tissues from these rats wereanalyzed for the presence of hCFTR protein by immunohistochemicalstaining.

Protein was detected throughout the entire lung, including the bronchialepithelial cells and the alveolar regions, as shown in FIG. 3. Positive(brown) staining was observed in the mRNA lipid nanoparticle testarticle group. Panel A depicts the CO-hCFTR mRNA ICE LNP-treated ratlung at a low magnification. Panel B depicts CO-hCFTR mRNA ICELNP-treated rat lung at 10× magnification. Panel C depicts CO-hCFTR mRNAICE LNP-treated rat lung at 20× magnification.

Example 4. In Vivo Expression of hCFTR in Rat Lungs (50 μg mRNA, 5% PEG)

FIG. 4 shows exemplary immunohistochemical analysis of hCFTR protein inrat lungs 24 hours after pulmonary delivery of CO-hCFTR mRNA lipidnanoparticles prepared by the process described above using ICE-basedlipid nanoparticles.

In some representative studies, rats were exposed to CO-hCFTR mRNA ICELNPs using direct instillation via a MicroSprayer apparatus. An aerosolwas produced via a MicroSprayer apparatus during intratrachealadministration of 50 μg CO-hCFTR mRNA encapsulated ICE LNPs (containing5% [mol] of PEG-modified lipid) in rats. The rats were then sacrificed24 hours after exposure and the lungs were harvested (a portion was snapfrozen and a separate portion was fixed in 10% NBF and embedded inparaffin) for analysis. The fixed lung tissues from these rats wereanalyzed for the presence of hCFTR protein by immunohistochemicalstaining.

Protein was detected throughout the entire lung, including the bronchialepithelial cells and the alveolar regions, as shown in FIG. 4. Positive(brown) staining was observed in the mRNA lipid nanoparticle testarticle group. Panel A depicts the CO-hCFTR mRNA ICE LNP-treated ratlung at a low magnification. Panel B depicts CO-hCFTR mRNA ICELNP-treated rat lung at 10× magnification. Panel C depicts CO-hCFTR mRNAICE LNP-treated rat lung at 20× magnification.

Example 5. In Vivo Expression of hCFTR in Rat Lungs (10 mg, 3% PEG)

FIG. 5 shows exemplary immunohistochemical analysis of hCFTR protein inrat lungs 24 hours after pulmonary delivery of CO-hCFTR mRNA lipidnanoparticles prepared by the process described above using ICE-basedlipid nanoparticles.

In some representative studies, rats were placed in an aerosol chamberwhich allowed for full motion of the rats. An aerosol was produced vianebulization of 10 mg CO-hCFTR mRNA encapsulated in ICE LNPs (containing3% [mol] of PEG-modified lipid) which filled the chamber containing therats. The rats were exposed for a given period of time during which theaerosol was freely taken into the lungs via normal breathing. After theexposure, the rats were removed and placed back into their cages. Therats were then sacrificed 24 hours after exposure and the lungs wereharvested (a portion was snap frozen and a separate portion was fixed in10% NBF and embedded in paraffin) for analysis. The fixed lung tissuesfrom these rats were analyzed for the presence of hCFTR protein byimmunohistochemical staining.

Protein was detected throughout the entire lung, including the bronchialepithelial cells and the alveolar regions, as shown in FIG. 5. Positive(brown) staining was observed in the mRNA lipid nanoparticle testarticle group. Panel A depicts the CO-hCFTR mRNA ICE LNP-treated ratlung at a low magnification. Panel B depicts CO-hCFTR mRNA ICELNP-treated rat lung at 10× magnification. Panel C depicts CO-hCFTR mRNAICE LNP-treated rat lung at 20× magnification.

Example 6. In Vivo Expression of hCFTR in Rat Lungs (50 μg mRNA, 3% PEG)

FIG. 6 shows exemplary immunohistochemical analysis of hCFTR protein inrat lungs 24 hours after pulmonary delivery of CO-hCFTR mRNA lipidnanoparticles prepared by the process described above using ICE-basedlipid nanoparticles.

In some representative studies, rats were exposed to CO-hCFTR mRNA ICELNPs using direct instillation via a MicroSprayer apparatus. An aerosolwas produced via a MicroSprayer apparatus during intratrachealadministration of 50 μg CO-hCFTR mRNA encapsulated ICE LNPs (containing3% [mol] of PEG-modified lipid) in rats. The rats were then sacrificed24 hours after exposure and the lungs were harvested (a portion was snapfrozen and a separate portion was fixed in 10% NBF and embedded inparaffin) for analysis. The fixed lung tissues from these rats wereanalyzed for the presence of hCFTR protein by immunohistochemicalstaining.

Protein was detected throughout the entire lung, including the bronchialepithelial cells and the alveolar regions, as shown in FIG. 6. Positive(brown) staining was observed in the mRNA lipid nanoparticle testarticle group. Panel A depicts the CO-hCFTR mRNA ICE LNP-treated ratlung at a low magnification. Panel B depicts CO-hCFTR mRNA ICELNP-treated rat lung at 10× magnification. Panel C depicts CO-hCFTR mRNAICE LNP-treated rat lung at 20× magnification.

Example 7. In Vivo Expression of hCFTR in Mouse Lungs (10 mg, N/P=2)

FIG. 7 shows exemplary immunohistochemical analysis of hCFTR protein inCFTR KO mouse lungs 24 hours after pulmonary delivery of CO-hCFTR mRNAlipid nanoparticles prepared by the process described above using ICEbased lipid nanoparticles.

In some representative studies, mice were placed in an aerosol chamberwhich allowed for full motion of the mice. An aerosol was produced vianebulization of 10 mg CO-hCFTR mRNA encapsulated in ICE LNPs (N/P=2)which filled the chamber containing the mice. The mice were exposed fora given period of time with which the aerosol was freely taken into thelungs via normal breathing. After the exposure, the mice were removedand placed back into their cages. The mice were then sacrificed 24 hoursafter exposure and the lungs were harvested (a portion was snap frozenand a separate portion was fixed in 10% NBF and embedded in paraffin)for analysis. The fixed lung tissues from these mice were analyzed forthe presence of hCFTR protein by immunohistochemical staining.

Protein was detected throughout the entire lung, including the bronchialepithelial cells and the alveolar regions, as shown in FIG. 7. Positive(brown) staining was observed in the mRNA lipid nanoparticle testarticle group. Panel A depicts the CO-hCFTR mRNA ICE LNP-treated mouselung at a low magnification. Panel B depicts CO-hCFTR mRNA ICELNP-treated mouse lung at 10× magnification. Panel C depicts CO-hCFTRmRNA ICE LNP-treated mouse lung at 20× magnification.

Example 8. In Vivo Expression of hCFTR in Mouse Lungs (10 mg, N/P=4)

FIG. 8 shows exemplary immunohistochemical analysis of hCFTR protein inCFTR KO mouse lungs 24 hours after pulmonary delivery of CO-hCFTR mRNAlipid nanoparticles prepared by the process described above usingICE-based lipid nanoparticles.

In some representative studies, mice were placed in an aerosol chamberwhich allowed for full motion of the mice. An aerosol was produced vianebulization of 10 mg CO-hCFTR mRNA encapsulated in ICE LNPs (N/P=4)which filled the chamber containing the mice. The mice were exposed fora given period of time with which the aerosol was freely taken into thelungs via normal breathing. After the exposure, the mice were removedand placed back into their cages. The mice were then sacrificed 24 hoursafter exposure and the lungs were harvested (a portion was snap frozenand a separate portion was fixed in 10% NBF and embedded in paraffin)for analysis. The fixed lung tissues from these mice were analyzed forthe presence of hCFTR protein by immunohistochemical staining.

Protein was detected throughout the entire lung, including the bronchialepithelial cells and the alveolar regions, as shown in FIG. 8. Positive(brown) staining was observed in the mRNA lipid nanoparticle testarticle group. Panel A depicts the CO-hCFTR mRNA ICE LNP-treated mouselung at a low magnification. Panel B depicts CO-hCFTR mRNA ICELNP-treated mouse lung at 10× magnification. Panel C depicts CO-hCFTRmRNA ICE LNP-treated mouse lung at 20× magnification. Widespreaddistribution of hCFTR protein is observed in both example 5 (N/P=2) andexample 6 (N/P=4).

Example 9. In Vivo Expression of hCFTR in Mouse Lungs with DifferentFormulations

FIG. 9 shows exemplary immunohistochemical analysis of hCFTR protein inwild-type mouse lungs 24 hours after pulmonary delivery of CO-hCFTR mRNAlipid nanoparticles prepared by the process described above usingICE-based lipid nanoparticles.

In some representative studies, several groups of wild-type mice wereseparated and placed in individual aerosol chambers. Each group wastreated with a different formulation. The formulations consisted ofICE-based LNPs (N/P=4) encapsulating the following mRNA constructs: 1)CO-hCFTR mRNA ICE LNP; 2) CO-hCFTR “STOP” mRNA (nonsense mutatedCO-hCFTR mRNA unable to translate protein) ICE LNP; 3) FFL (FireflyLuciferase) mRNA ICE LNP; and 4) Buffer (Vehicle).

In some embodiments, mice were placed in an aerosol chamber whichallowed for full motion of the mice. An aerosol was produced vianebulizer of CO-hCFTR mRNA encapsulated in ICE LNPs (N/P=4) which filledthe chamber containing the mice. The mice were exposed for a total of 50mg (shown in FIG. 7) aerosolized CO-hCFTR mRNA (as measured byencapsulated mRNA) for a given period of time with which the aerosol wasfreely taken into the lungs via normal breathing. After the exposure,the mice were removed and placed back into their cages. The mice werethen sacrificed 24 hours after exposure and the lungs were harvested (aportion was snap frozen and a separate portion was fixed in 10% NBF andembedded in paraffin) for analysis. The fixed lung tissues from thesemice were analyzed for the presence of hCFTR protein byimmunohistochemical staining.

In FIG. 9, positive (brown) staining was observed only in the CO-hCFTRmRNA LNP test article group. The first panel depicts the CO-hCFTR mRNAICE LNP-treated mouse lung showing widespread distribution of human CFTRprotein observed throughout the entire lung, including both bronchialepithelial and alveolar regions. The second panel depicts CO-hCFTR“STOP” mRNA ICE LNP-treated mouse lung with no positive (brown) stainingobserved. The third panel depicts FFL mRNA ICE LNP-treated mouse lungwith no positive (brown) staining observed. The fourth panel depictsbuffer treated mouse lung with no positive (brown) staining observed.

Example 10. In Vivo Expression of hCFTR in Mouse Lungs at Different TimePoints

FIG. 10 shows exemplary immunohistochemical analysis of hCFTR protein inwild-type mouse lungs at predetermined time points after pulmonarydelivery of CO-hCFTR mRNA lipid nanoparticles prepared by the processdescribed above using ICE-based lipid nanoparticles.

In some representative studies, the pharmacokinetic behavior of hCFTRprotein after treatment of CO-hCFTR mRNA encapsulated ICE LNPs wasperformed in CFTR KO mice. In some embodiments, mice were placed in anaerosol chamber which allowed for full motion of the mice. An aerosolwas produced via nebulization of CO-hCFTR mRNA encapsulated in ICE LNPs(N/P=4) which filled the chamber containing the mice. The mice wereexposed to 10 mg aerosolized CO-hCFTR mRNA for a given period of timeduring which the aerosol was freely taken into the lungs via normalbreathing. After the exposure, the mice were removed and placed backinto their cages. Selected cohorts of treated mice were sacrificed atpre-determined time points after aerosol exposure. The time range forsacrificing the mice post-administration ranged from 30 minutes to oneweek, specifically, 30 minutes, 2 hours, 4 hours, 6 hours, 24 hours, 48hours, 72 hours and 1 week as shown in FIG. 10. The mouse lungs wereharvested (a portion was snap frozen and a separate portion was fixed in10% NBF and embedded in paraffin) for analysis. The fixed lung tissuesfrom these mice were analyzed for the presence of hCFTR protein byimmunohistochemical staining.

As shown in FIG. 10, protein or positive (brown) staining was detectedthroughout the entire lung, including both the bronchial epithelialcells and the alveolar regions, in the hCFTR mRNA treated mouse groupssacrificed more than 30 minutes after administration. The most positiveor brown staining was observed at 24 hours, 48 hours and 72 hours afterhCFTR mRNA delivery. No positive (brown) staining was observed in thesaline treated control group.

Example 11. In Vivo Expression of hCFTR in Rat Lungs at Different DoseLevels

In some representative studies, rats were placed in an aerosol chamberwhich allowed for full motion of the rats. An aerosol was produced vianebulization of CO-hCFTR mRNA encapsulated in ICE LNPs which filled thechamber containing the rats. The rats were exposed for a given period oftime with which the aerosol was freely taken into the lungs via normalbreathing. Several groups were dosed at different dose levels asfollowing: 1) Buffer; 2) Empty ICE LNP (˜8.0 mg/kg equivalent); 3) ICELNP (0.5 mg/kg); 4) ICE LNP (2.1 mg/kg); 5) ICE LNP (4.1 mg/kg); and 6)ICE LNP (6.2 mg/kg).

After administration, the rats were removed and placed back into theircages. Furthermore, each group of rats was split into selected cohortsthat were sacrificed at different predetermined time pointspost-administration. The time range for sacrificing the ratspost-administration was 24 hours to 28 days, specifically 24 hours, 7days, 14 days and 28 days.

Quantitative PCR, which measures copies of CO-hCFTR mRNA per total RNA,was performed on snap frozen lungs of all treated rats in each group.The results are represented in FIG. 11, showing the ratio of copies ofCO-hCFTR mRNA per micrograms of total RNA. Results were analyzed acrossthe different doses administered and at the selected sacrifice timepoints for each dose level. Background levels of CO-hCFTR mRNA areindicated by the control groups' (Buffer and Empty LNP) values. Copiesof CO-hCFTR mRNA per total RNA in frozen lung sections of rats increasedas the dose level increased. At the same dose level, copies of CO-hCFTRmRNA per total RNA in frozen lung sections of rats decreased as thesacrifice time post-administration increased.

FIG. 12 shows a comparison of the levels of copies of exogenous CO-hCFTRmRNA per copy of endogenous CFTR mRNA. The fold-increase or ratio ofcopies of CO-hCFTR mRNA per copy of endogenous CFTR mRNA in frozen lungsections of rats was assessed and plotted as a function of dose andsacrifice time. The data was also obtained using Quantitative PCR.Fold-increase of copies of CO-hCFTR mRNA over endogenous levels of CFTRmRNA increased as the dose level increased. At the same mRNA dosage,fold-increase of copies of CO-hCFTR mRNA over endogenous levels of CFTRmRNA decreased as the sacrifice time post-administration increased.

Immunohistochemical analysis of the rat lungs was also performed and isshown in FIG. 13. The fixed lung tissues from these rats were analyzedfor the presence of hCFTR protein by immunohistochemical staining. Adose-dependent increase in hCFTR protein was observed as determined bypositive (brown) staining throughout the entire lung, including bothbronchial epithelial and alveolar regions. A decrease in hCFTR proteinwas observed as determined by positive (brown) staining as the timeafter the single dose of administration increased beyond 24 hours.However, detectable levels of hCFTR protein were observed 28 days aftera single exposure of hCFTR mRNA ICE LNPs. No positive (brown) stainingwas observed in the saline treated control group or in the empty LNPgroup.

Example 12. In Vivo Effect of hCFTR Formulations on Respiration

In some representative studies, rats were exposed to a single dose ofhCFTR mRNA via inhalation. The hCFTR mRNA was formulated with either ICEas described above or with branched PEI (bPEI). As shown in FIG. 14A,the various dosages of hCFTR mRNA formulated with bPEI caused an adverserespiratory rate increase relative to a saline control in the ratsstudied. When the rats were sacrificed, it was also found that theweight of the lungs was also increased. By comparison, as shown in FIG.14B, the various dosages of hCFTR mRNA formulated with ICE did not causean increase in respiratory rate relative to a buffer control in the ratsstudied. There was also no increase in the weight of the rat lungsobserved when the rats were sacrificed.

Example 13. In Vitro Expression and Activity of hCFTR at Different DoseLevels

In some representative experiments, hCFTR mRNA was transfected intocultured Fischer rat thyroid cells using Lipofectamine according tostandard procedures. As is shown in FIG. 15, there was a dose responseof chloride-ion channel activity induced by hCFTR mRNA. This indicatesthat the hCFTR mRNA produced active CFTR protein in the transfectedcells.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

We claim:
 1. A composition comprising: nucleic acids; and lipidnanoparticles encapsulating the nucleic acids, wherein each individuallipid nanoparticle comprises no more than three distinct lipidcomponents, one distinct lipid component being a sterol-based cationiclipid comprising imidazole cholesterol ester (ICE), wherein thesterol-based cationic lipid constitutes less than 70% molar ratio of thetotal lipids, and further wherein the lipid nanoparticles have anencapsulation percentage for nucleic acids of at least 70%.
 2. Thecomposition of claim 1, wherein the nucleic acids comprise mRNA encodinga Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein;and lipid nanoparticles encapsulating the mRNA, wherein each individuallipid nanoparticle comprises no more than three distinct lipidcomponents, one distinct lipid component being a sterol-based cationiclipid, and wherein the lipid nanoparticles have an encapsulationpercentage for mRNA of at least 80%.
 3. The composition of claim 2,wherein the mRNA encoding a CFTR protein comprises SEQ ID NO: 1, or apolynucleotide sequence at least 70%, 75%, 80%, 85%, 90%, or 95%identical to SEQ ID NO:
 1. 4. A method of delivering mRNA in vivocomprising administering a composition of claim 2 to a subject, whereinthe composition is administered intravenously, or by pulmonary delivery,wherein the pulmonary delivery comprises nebulization.
 5. The method ofclaim 1, wherein the nucleic acids are selected from DNA, siRNA,microRNA, and mRNA.
 6. The composition of claim 1, wherein the nucleicacids are mRNA encoding a protein or a peptide, wherein the mRNA iscodon-optimized, and comprises one or more modified nucleotides.